gtf gene  (New England Biolabs)


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    Name:
    Phusion High Fidelity DNA Polymerase
    Description:
    Phusion High Fidelity DNA Polymerase 500 units
    Catalog Number:
    m0530l
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    446
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    500 units
    Category:
    Thermostable DNA Polymerases
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    New England Biolabs gtf gene
    Phusion High Fidelity DNA Polymerase
    Phusion High Fidelity DNA Polymerase 500 units
    https://www.bioz.com/result/gtf gene/product/New England Biolabs
    Average 92 stars, based on 7230 article reviews
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    gtf gene - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Characterization of Pediococcus ethanolidurans CUPV141: A β-D-glucan- and Heteropolysaccharide-Producing Bacterium"

    Article Title: Characterization of Pediococcus ethanolidurans CUPV141: A β-D-glucan- and Heteropolysaccharide-Producing Bacterium

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02041

    Detection of plasmids of P. ethanolidurans CUPV141 and CUPV141NR strains and of P. parvulus 2.6. (A) Detection of the gtf gene by Southern blot hybridization. Left, analysis in a 0.7% agarose gel of plasmids preparations of LAB strains and of E. coli V517. Right, hybridized membrane of samples transferred from the agarose gel. (B) Depicts the calibration curve for plasmid size determination. Symbols: plasmids from E. coli V517 (◊), P. ethanolidurans (♦) and P. parvulus (♦) strains. (C) Analysis in 0.7% agarose gel of gtf PCR amplicons obtained with genomic DNA from CUPV141 and CUPV141NR strains. Smart Ladder, molecular weight standard.
    Figure Legend Snippet: Detection of plasmids of P. ethanolidurans CUPV141 and CUPV141NR strains and of P. parvulus 2.6. (A) Detection of the gtf gene by Southern blot hybridization. Left, analysis in a 0.7% agarose gel of plasmids preparations of LAB strains and of E. coli V517. Right, hybridized membrane of samples transferred from the agarose gel. (B) Depicts the calibration curve for plasmid size determination. Symbols: plasmids from E. coli V517 (◊), P. ethanolidurans (♦) and P. parvulus (♦) strains. (C) Analysis in 0.7% agarose gel of gtf PCR amplicons obtained with genomic DNA from CUPV141 and CUPV141NR strains. Smart Ladder, molecular weight standard.

    Techniques Used: Southern Blot, Hybridization, Agarose Gel Electrophoresis, Plasmid Preparation, Polymerase Chain Reaction, Molecular Weight

    2) Product Images from "Negative regulation of conserved RSL class I bHLH transcription factors evolved independently among land plants"

    Article Title: Negative regulation of conserved RSL class I bHLH transcription factors evolved independently among land plants

    Journal: eLife

    doi: 10.7554/eLife.38529

    MpFRH1 miRNA-resistant version of Mp RSL1 (Mp RSL1 res ) suppresses the Mp FRH1 GOF 2 few rhizoids and few gemmae phenotype. ( A ) Sequence of the MpFRH1 miRNA and its target site on the wild type Mp RSL1 mRNA. The cleavage site is indicated in red. ( B ) Point mutations introduced into the MpFRH1 miRNA target site on Mp RSL1 mRNA to create MpFRH1 miRNA resistant version of Mp RSL1 (Mp RSL1 res ). Mutated bases are indicated in red. ( C ) Examples phenotypes observed in Mp FRH1 GOF2 plants transformed with pro EF1a:MpRSL1 WT or pro EF1a:MpRSL1 res . Mature transformant plants, Scale bar 2 mm. ( D ) Frequencies of each phenotype observed in Mp FRH1 GOF2 plants transformed with either pro EF1a:MpRSL1 WT or pro EF1a:MpRSL1 res .
    Figure Legend Snippet: MpFRH1 miRNA-resistant version of Mp RSL1 (Mp RSL1 res ) suppresses the Mp FRH1 GOF 2 few rhizoids and few gemmae phenotype. ( A ) Sequence of the MpFRH1 miRNA and its target site on the wild type Mp RSL1 mRNA. The cleavage site is indicated in red. ( B ) Point mutations introduced into the MpFRH1 miRNA target site on Mp RSL1 mRNA to create MpFRH1 miRNA resistant version of Mp RSL1 (Mp RSL1 res ). Mutated bases are indicated in red. ( C ) Examples phenotypes observed in Mp FRH1 GOF2 plants transformed with pro EF1a:MpRSL1 WT or pro EF1a:MpRSL1 res . Mature transformant plants, Scale bar 2 mm. ( D ) Frequencies of each phenotype observed in Mp FRH1 GOF2 plants transformed with either pro EF1a:MpRSL1 WT or pro EF1a:MpRSL1 res .

    Techniques Used: Sequencing, Transformation Assay

    Transcripts of one of the four predicted MpFRH1 miRNA targets are less abundant in Mp FRH1 GOF2 compared to wild type. ( A ) RT-PCR quantification of transcript levels of the four predicted FRH1 miRNA target mRNAs in WT and Mp FRH1 GOF2 plants. ( B ) qRT-PCR quantification of transcript levels of Mp RSL1 . Mp RSL1 transcript levels were normalised against Mp APT1 and Mp CUL3 .
    Figure Legend Snippet: Transcripts of one of the four predicted MpFRH1 miRNA targets are less abundant in Mp FRH1 GOF2 compared to wild type. ( A ) RT-PCR quantification of transcript levels of the four predicted FRH1 miRNA target mRNAs in WT and Mp FRH1 GOF2 plants. ( B ) qRT-PCR quantification of transcript levels of Mp RSL1 . Mp RSL1 transcript levels were normalised against Mp APT1 and Mp CUL3 .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    Stem-loop PCR detection of the MpFRH1 miRNA. RNA extracted from 15 day old wild type and Mp FRH1 GOF1-3 gemmae was used as a template for the stem-loop PCR amplification. Amplification products after 28 PCR cycles were visualised on a EtBr stained 2% agarose gel. The tRNA band was used as a loading control.
    Figure Legend Snippet: Stem-loop PCR detection of the MpFRH1 miRNA. RNA extracted from 15 day old wild type and Mp FRH1 GOF1-3 gemmae was used as a template for the stem-loop PCR amplification. Amplification products after 28 PCR cycles were visualised on a EtBr stained 2% agarose gel. The tRNA band was used as a loading control.

    Techniques Used: Polymerase Chain Reaction, Amplification, Staining, Agarose Gel Electrophoresis

    The MpFRH1 miRNA target site is conserved in liverwort RSL class I transcripts. Liverwort RSL class I transcript alignment. The MpFRH1 miRNA target site is circled in red. Black columns indicate 100% conservation. Supplemental data file legends.
    Figure Legend Snippet: The MpFRH1 miRNA target site is conserved in liverwort RSL class I transcripts. Liverwort RSL class I transcript alignment. The MpFRH1 miRNA target site is circled in red. Black columns indicate 100% conservation. Supplemental data file legends.

    Techniques Used:

    Predicted targets of MpFRH1 miRNA. Lower scores indicate a more likely target based on sequence similarity between the miRNA and the target transcript. Transcript number corresponds to the M. polymorpha gametophyte transcriptome ( Honkanen et al., 2016 ). Mapoly gene ID correspond to the M. polymorpha genome sequence published in Bowman et al. (2017 ).
    Figure Legend Snippet: Predicted targets of MpFRH1 miRNA. Lower scores indicate a more likely target based on sequence similarity between the miRNA and the target transcript. Transcript number corresponds to the M. polymorpha gametophyte transcriptome ( Honkanen et al., 2016 ). Mapoly gene ID correspond to the M. polymorpha genome sequence published in Bowman et al. (2017 ).

    Techniques Used: Sequencing

    3) Product Images from "Enhanced Control of Oncolytic Measles Virus Using MicroRNA Target Sites"

    Article Title: Enhanced Control of Oncolytic Measles Virus Using MicroRNA Target Sites

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2018.04.002

    Mechanism of microRNA-Based Vector Control To elucidate the mechanism of microRNA-based vector control, a 3′ RACE (rapid amplification of cDNA ends) followed by sequencing approach was applied. Vero cells were transfected with miR-7-5p mimics and subsequently infected with MV-EGFP-H miRTS7 or MV-EGFP-H miRTS7rc at an MOI of 0.03. Thirty-five hours p.i., total RNA was isolated. (A) Schematic depiction of the RACE procedure. Total RNA was poly(A) tailed, and cDNA synthesis was performed using a RACE anchor primer. The RACE anchor primer contains a poly-T sequence complementary to the poly(A) tail of poly(A)-tailed RNAs, which is preceded by a nucleotide other than T (V) in order to position the primer at the beginning of the poly(A) sequence of the template. Two reactions of a nested PCR with primers complementary to regions upstream of the miRTS box (Pri 1, Pri 2) and regions within the RACE anchor sequence (RACE-I, RACE-O) were performed. Products of the second reaction of the nested PCR were then subjected to gel electrophoresis and bands containing PCR products of interest were cloned for subsequent Sanger sequencing. (B) Gel electrophoresis image showing products of the second reaction of the nested PCR. PCR products from samples transfected with miR-7-5p and infected with MV-EGFP-H miRTS7 (red box) were gel purified, cloned, and subjected to sequencing. (C) Sequencing result of cloned RACE-PCR fragments from H mRNA containing a miRTS7 box in presence of miR-7-5p. The upper sequence is the consensus cleavage sequence of the nine RACE-PCR fragments that showed cleavage within the first miRTS. The lower sequence shows the uncleaved sequence of the miRTS for miR-7-5p for comparison. Nucleotides are numbered from 3′ to 5′ starting at the 3′ end of the first microRNA target sequence with the miR-7-5p seed sequence indicated by a red box.
    Figure Legend Snippet: Mechanism of microRNA-Based Vector Control To elucidate the mechanism of microRNA-based vector control, a 3′ RACE (rapid amplification of cDNA ends) followed by sequencing approach was applied. Vero cells were transfected with miR-7-5p mimics and subsequently infected with MV-EGFP-H miRTS7 or MV-EGFP-H miRTS7rc at an MOI of 0.03. Thirty-five hours p.i., total RNA was isolated. (A) Schematic depiction of the RACE procedure. Total RNA was poly(A) tailed, and cDNA synthesis was performed using a RACE anchor primer. The RACE anchor primer contains a poly-T sequence complementary to the poly(A) tail of poly(A)-tailed RNAs, which is preceded by a nucleotide other than T (V) in order to position the primer at the beginning of the poly(A) sequence of the template. Two reactions of a nested PCR with primers complementary to regions upstream of the miRTS box (Pri 1, Pri 2) and regions within the RACE anchor sequence (RACE-I, RACE-O) were performed. Products of the second reaction of the nested PCR were then subjected to gel electrophoresis and bands containing PCR products of interest were cloned for subsequent Sanger sequencing. (B) Gel electrophoresis image showing products of the second reaction of the nested PCR. PCR products from samples transfected with miR-7-5p and infected with MV-EGFP-H miRTS7 (red box) were gel purified, cloned, and subjected to sequencing. (C) Sequencing result of cloned RACE-PCR fragments from H mRNA containing a miRTS7 box in presence of miR-7-5p. The upper sequence is the consensus cleavage sequence of the nine RACE-PCR fragments that showed cleavage within the first miRTS. The lower sequence shows the uncleaved sequence of the miRTS for miR-7-5p for comparison. Nucleotides are numbered from 3′ to 5′ starting at the 3′ end of the first microRNA target sequence with the miR-7-5p seed sequence indicated by a red box.

    Techniques Used: Plasmid Preparation, Rapid Amplification of cDNA Ends, Sequencing, Transfection, Infection, Isolation, Nested PCR, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Clone Assay, Purification

    Functional Analysis and Comparison of Different miRTS Box Positions Vero cells were transfected with 0 or 40 nM miR-7-5p mimics and subsequently infected with MV-EGFP-N miRTS7 /-F miRTS7 /-H miRTS7 /-L miRTS7 or MV-EGFP at an MOI of 0.03. (A) Fluorescence and phase contrast microscopy images 34 hr post-infection, ×50 magnification. (B) Progeny virus determination. Thirty-six hours post-infection, cells were scraped into their medium. Virus progeny titers were determined and are shown with normalization to progeny virus titers in absence of transfected microRNA. Error bars represent SD of three technical replicates per sample. (C) Cell viability of all samples was determined at 96 hr post-infection using a colorimetric (XTT) assay. Error bars represent SD of three technical replicates per sample. (D) Total RNA was isolated 32 hr post infection, subjected to RT-PCR, and cDNA was used for PCR. Gene-specific primer pairs corresponding to regions up- and downstream of the miRTS box insertion sites within the N , F , H , and L ORF, respectively, were used (upper gel), while β-actin-specific primers were used as an input control (lower gel). RT-PCR products were subjected to agarose gel electrophoresis.
    Figure Legend Snippet: Functional Analysis and Comparison of Different miRTS Box Positions Vero cells were transfected with 0 or 40 nM miR-7-5p mimics and subsequently infected with MV-EGFP-N miRTS7 /-F miRTS7 /-H miRTS7 /-L miRTS7 or MV-EGFP at an MOI of 0.03. (A) Fluorescence and phase contrast microscopy images 34 hr post-infection, ×50 magnification. (B) Progeny virus determination. Thirty-six hours post-infection, cells were scraped into their medium. Virus progeny titers were determined and are shown with normalization to progeny virus titers in absence of transfected microRNA. Error bars represent SD of three technical replicates per sample. (C) Cell viability of all samples was determined at 96 hr post-infection using a colorimetric (XTT) assay. Error bars represent SD of three technical replicates per sample. (D) Total RNA was isolated 32 hr post infection, subjected to RT-PCR, and cDNA was used for PCR. Gene-specific primer pairs corresponding to regions up- and downstream of the miRTS box insertion sites within the N , F , H , and L ORF, respectively, were used (upper gel), while β-actin-specific primers were used as an input control (lower gel). RT-PCR products were subjected to agarose gel electrophoresis.

    Techniques Used: Functional Assay, Transfection, Infection, Fluorescence, Microscopy, XTT Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Genome Structure of Recombinant Measles Viruses (A) Genome structure of engineered viruses encoding EGFP (enhanced GFP) in an additional transcription unit (ATU) upstream of the N gene as well as a synthetic microRNA target site (miRTS) box in the 3′ UTR of the N , F , H , or L gene. (B) Genome structure of engineered viruses encoding EGFP (enhanced GFP) in an additional transcription unit (ATU) upstream of the N gene as well as synthetic miRTS boxes in the 3′ UTRs of the N and F or N and L genes. (C) Sequences of four different miRTS boxes complementary to miR-7-5p, miR-122-5p, miR-124-3p, and miR-125b-5p, respectively. miRTS are underlined and separated by nonunderlined spacer nucleotides. The miRTS portions that are complementary to the microRNA seed sequences are indicated in bold print.
    Figure Legend Snippet: Genome Structure of Recombinant Measles Viruses (A) Genome structure of engineered viruses encoding EGFP (enhanced GFP) in an additional transcription unit (ATU) upstream of the N gene as well as a synthetic microRNA target site (miRTS) box in the 3′ UTR of the N , F , H , or L gene. (B) Genome structure of engineered viruses encoding EGFP (enhanced GFP) in an additional transcription unit (ATU) upstream of the N gene as well as synthetic miRTS boxes in the 3′ UTRs of the N and F or N and L genes. (C) Sequences of four different miRTS boxes complementary to miR-7-5p, miR-122-5p, miR-124-3p, and miR-125b-5p, respectively. miRTS are underlined and separated by nonunderlined spacer nucleotides. The miRTS portions that are complementary to the microRNA seed sequences are indicated in bold print.

    Techniques Used: Recombinant

    4) Product Images from "CRISPR-induced RASGAP deficiencies in colorectal cancer organoids reveal that only loss of NF1 promotes resistance to EGFR inhibition"

    Article Title: CRISPR-induced RASGAP deficiencies in colorectal cancer organoids reveal that only loss of NF1 promotes resistance to EGFR inhibition

    Journal: Oncotarget

    doi: 10.18632/oncotarget.26677

    Puromycin selected NF1 knock out CRC organoids show enhanced RAS and ERK activation ( A ) In comparison to P18T and P18T RASA1 KO (clone # 3), predominantly P18T NF1 KO (clone # 12) organoids show enhanced basal and reactivated ERK phosphorylation levels after 24 hr treatment with CRC medium containing 1 μM afatinib. Representative from n = 3 independent experiments. ( B ) NF1-deficient organoids (clone # 6 and # 12) show residual ERK phosphorylation after treatment with CRC medium containing 1 μM afatinib with varying kinetics. ( C ) Loss of NF1 (clone # 12) leads to elevated levels of RAS activity (GTP-loading) at basal conditions compared to P18T and P18T RASA1 KO (clone # 3) CRC organoids. The presence of an oncogenic mutation in KRAS (P18T KRAS G12D ) leads to elevated and sustained high levels of RAS activity (GTP-loading) at basal and in afatinib-treated conditions, respectively. RAS immunoblots from RAS pull-down assays are shown (RAS-GTP), together with a RAS immunoblot from total cell lysates as loading control. HRAS, KRAS, and NRAS isoforms are detected. Representative from n = 2 independent experiments. ( D ) Immunoblots of P18T, P18T RASA1 KO (clone # 1 and 3), P18T NF1 KO (clone # 6 and 12) CRC organoids indicate that the loss of RASA1 does not result in elevated protein levels of NF1, and vice versa. Representative from n = 3 independent experiments. ( E ) The relative expression levels of indicated RASGAPs genes that contain an active GAP domain were analyzed in P18T, P18T RASA1 KO (clone # 1 and 3), P18T NF1 KO (clone # 6 and 12) CRC organoids using RT-PCR. The relative expression of each RASGAP gene was normalized to the B2M housekeeping gene (representative from n = 3 independent experiments).
    Figure Legend Snippet: Puromycin selected NF1 knock out CRC organoids show enhanced RAS and ERK activation ( A ) In comparison to P18T and P18T RASA1 KO (clone # 3), predominantly P18T NF1 KO (clone # 12) organoids show enhanced basal and reactivated ERK phosphorylation levels after 24 hr treatment with CRC medium containing 1 μM afatinib. Representative from n = 3 independent experiments. ( B ) NF1-deficient organoids (clone # 6 and # 12) show residual ERK phosphorylation after treatment with CRC medium containing 1 μM afatinib with varying kinetics. ( C ) Loss of NF1 (clone # 12) leads to elevated levels of RAS activity (GTP-loading) at basal conditions compared to P18T and P18T RASA1 KO (clone # 3) CRC organoids. The presence of an oncogenic mutation in KRAS (P18T KRAS G12D ) leads to elevated and sustained high levels of RAS activity (GTP-loading) at basal and in afatinib-treated conditions, respectively. RAS immunoblots from RAS pull-down assays are shown (RAS-GTP), together with a RAS immunoblot from total cell lysates as loading control. HRAS, KRAS, and NRAS isoforms are detected. Representative from n = 2 independent experiments. ( D ) Immunoblots of P18T, P18T RASA1 KO (clone # 1 and 3), P18T NF1 KO (clone # 6 and 12) CRC organoids indicate that the loss of RASA1 does not result in elevated protein levels of NF1, and vice versa. Representative from n = 3 independent experiments. ( E ) The relative expression levels of indicated RASGAPs genes that contain an active GAP domain were analyzed in P18T, P18T RASA1 KO (clone # 1 and 3), P18T NF1 KO (clone # 6 and 12) CRC organoids using RT-PCR. The relative expression of each RASGAP gene was normalized to the B2M housekeeping gene (representative from n = 3 independent experiments).

    Techniques Used: Knock-Out, Activation Assay, Activity Assay, Mutagenesis, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

    CRISPR screen against RASGAPs in patient-derived CRC organoids reveals increased growth and EGF-independent survival upon loss of NF1 GAP activity ( A ) The mRNA expression level of 9 RASGAPs containing an active GAP domain was analyzed in P18T organoids using qPCR. The relative expression of each RASGAP gene was normalized to the B2M housekeeping gene (representative from n = 3 independent experiments). ( B ) Left; schematic representation of expression plasmid containing both an U6 promoter-driven sgRNA and a CBh promoter-driven SpCas9-2A-GFP was used to target the RASGAP domain. Right; schematic overview of the RASGAP knock out screen in P18T patient-derived CRC organoids that are wild type for the RAS signaling pathway. ( C ) P18T CRC organoids in selection medium that have been transfected with indicated sgRNAs and Cas9. White arrow heads indicate representative background organoids. Yellow arrows indicate successful organoids that are significantly larger than background. Bar graph depicts the relative number of organoids with a size larger than background organoids as determined in the negative control. Area of alive RASGAP knock out organoids was measured using calcein green assay (see Materials and Methods).
    Figure Legend Snippet: CRISPR screen against RASGAPs in patient-derived CRC organoids reveals increased growth and EGF-independent survival upon loss of NF1 GAP activity ( A ) The mRNA expression level of 9 RASGAPs containing an active GAP domain was analyzed in P18T organoids using qPCR. The relative expression of each RASGAP gene was normalized to the B2M housekeeping gene (representative from n = 3 independent experiments). ( B ) Left; schematic representation of expression plasmid containing both an U6 promoter-driven sgRNA and a CBh promoter-driven SpCas9-2A-GFP was used to target the RASGAP domain. Right; schematic overview of the RASGAP knock out screen in P18T patient-derived CRC organoids that are wild type for the RAS signaling pathway. ( C ) P18T CRC organoids in selection medium that have been transfected with indicated sgRNAs and Cas9. White arrow heads indicate representative background organoids. Yellow arrows indicate successful organoids that are significantly larger than background. Bar graph depicts the relative number of organoids with a size larger than background organoids as determined in the negative control. Area of alive RASGAP knock out organoids was measured using calcein green assay (see Materials and Methods).

    Techniques Used: CRISPR, Derivative Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Knock-Out, Selection, Transfection, Negative Control

    Puromycin selected NF1 knock out CRC organoids show insensitivity to EGFR inhibition ( A ) A schematic overview illustrating the strategy to score sensitivity of NF1 and RASA1 knock out organoids of similar size treated with colorectal cancer (CRC) medium containing either DMSO or 1 μM afatinib (EGFRi) for 72 hours. ( B ) Representative pictures of the parental patient-derived CRC organoids P18T and P18T RASA1 (clone # 1 and # 3) or NF1 (# 6 and # 12) knock out organoids prior (day 0) and after 72 hours of DMSO or 1 μM afatinib treatment (Day 3). White asterisks indicate dead organoids. Scale bars, 100 μM. Bar graph depicts the percentage of living organoids (out of 100 organoid counts) based on morphology.
    Figure Legend Snippet: Puromycin selected NF1 knock out CRC organoids show insensitivity to EGFR inhibition ( A ) A schematic overview illustrating the strategy to score sensitivity of NF1 and RASA1 knock out organoids of similar size treated with colorectal cancer (CRC) medium containing either DMSO or 1 μM afatinib (EGFRi) for 72 hours. ( B ) Representative pictures of the parental patient-derived CRC organoids P18T and P18T RASA1 (clone # 1 and # 3) or NF1 (# 6 and # 12) knock out organoids prior (day 0) and after 72 hours of DMSO or 1 μM afatinib treatment (Day 3). White asterisks indicate dead organoids. Scale bars, 100 μM. Bar graph depicts the percentage of living organoids (out of 100 organoid counts) based on morphology.

    Techniques Used: Knock-Out, Inhibition, Derivative Assay

    Puromycin selected NF1 knock out CRC organoids show enhanced organoid growth upon release of RAS-MAPK pathway inhibition ( A ) A schematic overview illustrating the strategy to score sensitivity and outgrowth of P18T parental, RASGAP knock out, and oncogenic mutant KRAS organoids of similar size during and after treatment with colorectal cancer (CRC) medium containing either DMSO, 1 μM afatinib (EGFRi), 1 μM selumetinib (MEKi), or a combination of 1 μM afatinib and 1 μM selumetinib. Organoid size and frequency of alive organoids was quantified after 72 hr of drug treatment and after 7 days of drug withdrawal by phenotypic analysis. ( B ) Representative zoom-in pictures of the parental patient-derived CRC organoids P18T KRAS WT , KRAS G12D , and P18T RASA1 (clone # 1 and # 3) or NF1 (# 6 and # 12) after 72 hours of DMSO or targeted drug treatment (on treatment). ( C ) Representative zoom-in pictures of the parental patient-derived CRC organoids P18T KRAS WT , KRAS G12D , and P18T RASA1 (clone # 1 and # 3) or NF1 (# 6 and # 12) after 7 days of DMSO or drug withdrawal (off treatment). Hoechst and DRAQ7 was used to visualize nuclei and dead cells, respectively.
    Figure Legend Snippet: Puromycin selected NF1 knock out CRC organoids show enhanced organoid growth upon release of RAS-MAPK pathway inhibition ( A ) A schematic overview illustrating the strategy to score sensitivity and outgrowth of P18T parental, RASGAP knock out, and oncogenic mutant KRAS organoids of similar size during and after treatment with colorectal cancer (CRC) medium containing either DMSO, 1 μM afatinib (EGFRi), 1 μM selumetinib (MEKi), or a combination of 1 μM afatinib and 1 μM selumetinib. Organoid size and frequency of alive organoids was quantified after 72 hr of drug treatment and after 7 days of drug withdrawal by phenotypic analysis. ( B ) Representative zoom-in pictures of the parental patient-derived CRC organoids P18T KRAS WT , KRAS G12D , and P18T RASA1 (clone # 1 and # 3) or NF1 (# 6 and # 12) after 72 hours of DMSO or targeted drug treatment (on treatment). ( C ) Representative zoom-in pictures of the parental patient-derived CRC organoids P18T KRAS WT , KRAS G12D , and P18T RASA1 (clone # 1 and # 3) or NF1 (# 6 and # 12) after 7 days of DMSO or drug withdrawal (off treatment). Hoechst and DRAQ7 was used to visualize nuclei and dead cells, respectively.

    Techniques Used: Knock-Out, Inhibition, Mutagenesis, Derivative Assay

    5) Product Images from "The transcription factor NHR-8: A new target to increase ivermectin efficacy in nematodes"

    Article Title: The transcription factor NHR-8: A new target to increase ivermectin efficacy in nematodes

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007598

    Effects of loss of NHR-8 function on susceptibility of Caenorhabditis elegans to IVM. (A) Dose response curves to IVM in a larval development assay of nhr-8(ok186) in comparison to the wild-type Bristol N2. Values represent the percentage of L1 reaching the young adult stage after 55 hours of incubation at 21°C within the presence of increasing doses of IVM. Data are mean ± SEM from 6 independent experiments. (B) Microfluidic Electropharyngeograms (EPGs) recorded from wild-type N2 Bristol and nhr-8(ok186) with or without IVM exposure. (C) Analysis of pharyngeal pumping activity (pump frequency) showing hypersensitivity of nhr-8(ok186) mutant to IVM and the rescue of IVM sensitivity by C . elegans nhr-8 cDNA. Pump frequency was compared in worms exposed to control or 0.1 μM IVM using the Nemametrix ScreenChip system. EPG recordings (2–4 min per worm) were started 20 min after the onset of IVM exposure. Data are reported as the mean ± SEM; n = 30–90 worms/group. a p
    Figure Legend Snippet: Effects of loss of NHR-8 function on susceptibility of Caenorhabditis elegans to IVM. (A) Dose response curves to IVM in a larval development assay of nhr-8(ok186) in comparison to the wild-type Bristol N2. Values represent the percentage of L1 reaching the young adult stage after 55 hours of incubation at 21°C within the presence of increasing doses of IVM. Data are mean ± SEM from 6 independent experiments. (B) Microfluidic Electropharyngeograms (EPGs) recorded from wild-type N2 Bristol and nhr-8(ok186) with or without IVM exposure. (C) Analysis of pharyngeal pumping activity (pump frequency) showing hypersensitivity of nhr-8(ok186) mutant to IVM and the rescue of IVM sensitivity by C . elegans nhr-8 cDNA. Pump frequency was compared in worms exposed to control or 0.1 μM IVM using the Nemametrix ScreenChip system. EPG recordings (2–4 min per worm) were started 20 min after the onset of IVM exposure. Data are reported as the mean ± SEM; n = 30–90 worms/group. a p

    Techniques Used: Incubation, Activity Assay, Mutagenesis

    Impact of Hco-nhr-8 silencing on IVM efficacy against susceptible (HcS-Wey) and resistant (HcR-Kok) H . contortus isolates. Larval feeding inhibition assay was performed by treating L2 larvae with increasing concentration of IVM, with or without silencing of Hco-nhr-8 through RNAi technique using whether double stranded RNA specifically targeting Hco-nhr-8 or non-target ( gfp ) siRNA as a control. The LFI 50 estimates the concentration of IVM at which 50% of the L2 did not feed. Data are mean ± SEM from 3 independent experiments.
    Figure Legend Snippet: Impact of Hco-nhr-8 silencing on IVM efficacy against susceptible (HcS-Wey) and resistant (HcR-Kok) H . contortus isolates. Larval feeding inhibition assay was performed by treating L2 larvae with increasing concentration of IVM, with or without silencing of Hco-nhr-8 through RNAi technique using whether double stranded RNA specifically targeting Hco-nhr-8 or non-target ( gfp ) siRNA as a control. The LFI 50 estimates the concentration of IVM at which 50% of the L2 did not feed. Data are mean ± SEM from 3 independent experiments.

    Techniques Used: Inhibition, Concentration Assay

    Effects of nhr-8 silencing on susceptibilities of wild-type N2 Bristol (A), IVM-selected (IVR10) (B) and MOX-selected (C) C . elegans strains to IVM in a larval development assay. Worms were fed on HT115 bacteria transformed whether with L4440 vector that produces double-stranded RNA against the Cel-nhr-8 gene, or with the empty vector as control. Values for dose response curves to IVM of IVM- and MOX-selected worms, both IVM-resistant, and wild-type represent the percentage of L1 reaching the young adult stage after 55 hours of incubation at 21°C within the presence of increasing doses of IVM. Data are mean ± SD from 4–6 independent experiments. Efficiencies of nhr-8 knockdown are presented in S2 Fig and S2 Table .
    Figure Legend Snippet: Effects of nhr-8 silencing on susceptibilities of wild-type N2 Bristol (A), IVM-selected (IVR10) (B) and MOX-selected (C) C . elegans strains to IVM in a larval development assay. Worms were fed on HT115 bacteria transformed whether with L4440 vector that produces double-stranded RNA against the Cel-nhr-8 gene, or with the empty vector as control. Values for dose response curves to IVM of IVM- and MOX-selected worms, both IVM-resistant, and wild-type represent the percentage of L1 reaching the young adult stage after 55 hours of incubation at 21°C within the presence of increasing doses of IVM. Data are mean ± SD from 4–6 independent experiments. Efficiencies of nhr-8 knockdown are presented in S2 Fig and S2 Table .

    Techniques Used: Transformation Assay, Plasmid Preparation, Incubation

    Effect of nhr-8 loss on PGP function in C . elegans . PGP function was evaluated by measuring the accumulation of the fluorescent dye rhodamine 123 in C . elegans . Wild-type Bristol N2 and nhr-8(ok186) young adult worms were incubated with rho123 at 10 μM for 48 h at 21°C and then examined by fluorescence microscopy for fluorescence intensity quantification. (A) Representative micrographs of individual Bristol N2 and nhr-8(ok186) worms after a 48h incubation with rhodamine 123; (B) Quantification of fluorescence intensity in Bristol N2 and nhr-8(ok186) worms. Data are mean ± SEM from 30–40 worms per strain. *** p
    Figure Legend Snippet: Effect of nhr-8 loss on PGP function in C . elegans . PGP function was evaluated by measuring the accumulation of the fluorescent dye rhodamine 123 in C . elegans . Wild-type Bristol N2 and nhr-8(ok186) young adult worms were incubated with rho123 at 10 μM for 48 h at 21°C and then examined by fluorescence microscopy for fluorescence intensity quantification. (A) Representative micrographs of individual Bristol N2 and nhr-8(ok186) worms after a 48h incubation with rhodamine 123; (B) Quantification of fluorescence intensity in Bristol N2 and nhr-8(ok186) worms. Data are mean ± SEM from 30–40 worms per strain. *** p

    Techniques Used: Incubation, Fluorescence, Microscopy

    Time course of stepwise selection of IVM tolerance in wild-type and nhr-8 -deficient C . elegans strains. Comparison between the development kinetics of acquired resistance to IVM in wild-type N2 Bristol (solid line) and nhr-8 - (ok186) (dashed line) C . elegans strains following stepwise exposure to IVM. Worms, cultured on Nematode Growth Medium plates containing IVM, were transferred to NGM plates containing higher doses of MLs when able to survive and reproduce. The period of 35 weeks corresponds approximately to 60 generations for each strain. Data representative of three separate experiments.
    Figure Legend Snippet: Time course of stepwise selection of IVM tolerance in wild-type and nhr-8 -deficient C . elegans strains. Comparison between the development kinetics of acquired resistance to IVM in wild-type N2 Bristol (solid line) and nhr-8 - (ok186) (dashed line) C . elegans strains following stepwise exposure to IVM. Worms, cultured on Nematode Growth Medium plates containing IVM, were transferred to NGM plates containing higher doses of MLs when able to survive and reproduce. The period of 35 weeks corresponds approximately to 60 generations for each strain. Data representative of three separate experiments.

    Techniques Used: Selection, Cell Culture

    6) Product Images from "Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library"

    Article Title: Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37233-y

    KlenTaq DNA polymerase mutant with improved allelic discrimination. ( A ) Sequence of employed primer-template pair. The 3′ end of primer either terminates with a matched (G) or mismatched (A) nucleotide. Primer extension reaction, employing the indicated DNA substrates along with wild-type (WT) and mutant DNA polymerase Mut_ADL, respectively, are shown as indicated. Reactions were stopped at different time points, as indicated. Full-length gel is presented in Supplementary Fig. S13 ( B , C ) Real time PCR experiments with purified enzymes (100 nM), showing the discrimination property of ( B ) wild-type KlenTaq DNA polymerase and ( C ) mutant DNA polymerase Mut_ADL respectively.
    Figure Legend Snippet: KlenTaq DNA polymerase mutant with improved allelic discrimination. ( A ) Sequence of employed primer-template pair. The 3′ end of primer either terminates with a matched (G) or mismatched (A) nucleotide. Primer extension reaction, employing the indicated DNA substrates along with wild-type (WT) and mutant DNA polymerase Mut_ADL, respectively, are shown as indicated. Reactions were stopped at different time points, as indicated. Full-length gel is presented in Supplementary Fig. S13 ( B , C ) Real time PCR experiments with purified enzymes (100 nM), showing the discrimination property of ( B ) wild-type KlenTaq DNA polymerase and ( C ) mutant DNA polymerase Mut_ADL respectively.

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

    Allelic discrimination on genomic DNA context. Real time PCR experiments for the detection of single nucleotide variants within the olfactory receptor for wild-type KlenTaq DNA polymerase (WT) ( A ) and Mut_ADL ( B ) with HeLa genomic DNA. Match (solid) and mismatch (dashed) reactions shown in amplification plot. Inset graph, showing melting peaks for the products of respective PCR reactions of wild-type KlenTaq DNA polymerase and Mut_ADL. ( C ) Agarose gel electrophoresis, showing the PCR product (109 bp) of match (M) and mismatch (Ms) reactions of wild-type KlenTaq DNA polymerase and Mut_ADL, respectively. Full-length gel is presented in Supplementary Fig. S14 . ( D ) Mutations contributing to improved discrimination property are depicted in crystal structure of KlenTaq DNA polymerase (in red). ( E ) Gel showing the processivity of wild-type KlenTaq DNA polymerase (WT) and Mut_ADL performed in presence of heparin (0.25 mg/ml and 0.5 mg/ml). Full-length gel is presented in Supplementary Fig. S15 .
    Figure Legend Snippet: Allelic discrimination on genomic DNA context. Real time PCR experiments for the detection of single nucleotide variants within the olfactory receptor for wild-type KlenTaq DNA polymerase (WT) ( A ) and Mut_ADL ( B ) with HeLa genomic DNA. Match (solid) and mismatch (dashed) reactions shown in amplification plot. Inset graph, showing melting peaks for the products of respective PCR reactions of wild-type KlenTaq DNA polymerase and Mut_ADL. ( C ) Agarose gel electrophoresis, showing the PCR product (109 bp) of match (M) and mismatch (Ms) reactions of wild-type KlenTaq DNA polymerase and Mut_ADL, respectively. Full-length gel is presented in Supplementary Fig. S14 . ( D ) Mutations contributing to improved discrimination property are depicted in crystal structure of KlenTaq DNA polymerase (in red). ( E ) Gel showing the processivity of wild-type KlenTaq DNA polymerase (WT) and Mut_ADL performed in presence of heparin (0.25 mg/ml and 0.5 mg/ml). Full-length gel is presented in Supplementary Fig. S15 .

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Mass Spectrometry

    ( A ) Crystal structure of large fragment of DNA polymerase I (KlenTaq) from Thermus aquaticus (PDB ID 3KTQ) 23 . The N-terminal domain is highlighted in grey. The DNA complex containing primer (green) and template (yellow) is depicted. The finger, palm and thumb domains are depicted in cyan, red and magenta, respectively. ( B ) Rationally selected target sites for focused library construction are shown in the primary structure of KlenTaq DNA polymerase. The residues selected for mutations are highlighted in coloured boxes and the location of residues in different domains of KlenTaq DNA polymerase are shown in red (palm), magenta (thumb) and cyan (finger). Evolutionarily conserved residues are marked with asterisk sign on the top of amino acids. ( C – E ) Detailed view of rationally selected target residues from finger, palm and thumb domains, respectively. The incoming dideoxycytidine triphosphate is shown in dark blue. ( F ) Activity profile of target amino acid sites investigated by site-directed mutagenesis and denoted in their respective domain (finger, palm and thumb) colour code. Amino acid substitutions resulting in PCR active mutants are denoted in green (Cq 1–30) and the inactive mutants are shown in black (Cq > 36). Blue indicates mutants with reduced activity (Cq 31–35) and the parent amino acids are highlighted in grey. Circled numbers indicate the active mutants included for molecular shuffling in the combinatorial library.
    Figure Legend Snippet: ( A ) Crystal structure of large fragment of DNA polymerase I (KlenTaq) from Thermus aquaticus (PDB ID 3KTQ) 23 . The N-terminal domain is highlighted in grey. The DNA complex containing primer (green) and template (yellow) is depicted. The finger, palm and thumb domains are depicted in cyan, red and magenta, respectively. ( B ) Rationally selected target sites for focused library construction are shown in the primary structure of KlenTaq DNA polymerase. The residues selected for mutations are highlighted in coloured boxes and the location of residues in different domains of KlenTaq DNA polymerase are shown in red (palm), magenta (thumb) and cyan (finger). Evolutionarily conserved residues are marked with asterisk sign on the top of amino acids. ( C – E ) Detailed view of rationally selected target residues from finger, palm and thumb domains, respectively. The incoming dideoxycytidine triphosphate is shown in dark blue. ( F ) Activity profile of target amino acid sites investigated by site-directed mutagenesis and denoted in their respective domain (finger, palm and thumb) colour code. Amino acid substitutions resulting in PCR active mutants are denoted in green (Cq 1–30) and the inactive mutants are shown in black (Cq > 36). Blue indicates mutants with reduced activity (Cq 31–35) and the parent amino acids are highlighted in grey. Circled numbers indicate the active mutants included for molecular shuffling in the combinatorial library.

    Techniques Used: Activity Assay, Mutagenesis, Polymerase Chain Reaction

    Evolved KlenTaq DNA polymerase with reverse transcriptase activity. ( A ) Depiction of novel mutations contributing to reverse transcriptase activity marked in red. ( B ) Reverse transcriptase activity studied by primer extension experiments for WT and Mut_RT. Reactions carried out for the indicated time points under identical conditions. P = primer, C = Control reaction that was carried out with the corresponding DNA template. Full-length gel is presented in Supplementary Fig. S16 . ( C ) Reverse transcriptase activity of Mut_RT in real time PCR assay employed with varying amounts of RNA template (left) and their corresponding melting peak analysis (right). ( D ) Employability of Mut_RT in reverse transcription from total RNA extract (100 ng) using the HPRT mRNA target (left) and the agarose gel analysis of amplified product of HRPT transcript (lane 1) and the no template control (lane2) Full-length gel is presented in Supplementary Fig. S17 .
    Figure Legend Snippet: Evolved KlenTaq DNA polymerase with reverse transcriptase activity. ( A ) Depiction of novel mutations contributing to reverse transcriptase activity marked in red. ( B ) Reverse transcriptase activity studied by primer extension experiments for WT and Mut_RT. Reactions carried out for the indicated time points under identical conditions. P = primer, C = Control reaction that was carried out with the corresponding DNA template. Full-length gel is presented in Supplementary Fig. S16 . ( C ) Reverse transcriptase activity of Mut_RT in real time PCR assay employed with varying amounts of RNA template (left) and their corresponding melting peak analysis (right). ( D ) Employability of Mut_RT in reverse transcription from total RNA extract (100 ng) using the HPRT mRNA target (left) and the agarose gel analysis of amplified product of HRPT transcript (lane 1) and the no template control (lane2) Full-length gel is presented in Supplementary Fig. S17 .

    Techniques Used: Activity Assay, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification

    7) Product Images from "Efficiency and precision of microRNA biogenesis modes in plants"

    Article Title: Efficiency and precision of microRNA biogenesis modes in plants

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky853

    Detection of miRNA processing intermediates in wild-type plants. ( A ) Scheme of the SPARE method (see Supplementary Figure S2 for details). MIRNAs are cut by DCL1 (grey triangles) and the resulting fragments with free 5′ end are ligated to an RNA oligo (purple line) and subjected to retro-transcription (green arrow). DNA is sequenced after PCR amplification. (B–E) Predicted secondary structure of ( B ) MIR173 , ( C ) MIR399a , ( D ) MIR399b , and ( E ) MIR399c . The miRNA is indicated in red and the miRNA* in light purple. Horizontal lines indicate cleavage sites detected in wild-type plants (green) and fiery1 mutants (gray). Independent reads for each cut are shown as numbers next to the lines. A green box highlights a 15–17 bp dsRNA segment below the miRNA/miRNA* duplex.
    Figure Legend Snippet: Detection of miRNA processing intermediates in wild-type plants. ( A ) Scheme of the SPARE method (see Supplementary Figure S2 for details). MIRNAs are cut by DCL1 (grey triangles) and the resulting fragments with free 5′ end are ligated to an RNA oligo (purple line) and subjected to retro-transcription (green arrow). DNA is sequenced after PCR amplification. (B–E) Predicted secondary structure of ( B ) MIR173 , ( C ) MIR399a , ( D ) MIR399b , and ( E ) MIR399c . The miRNA is indicated in red and the miRNA* in light purple. Horizontal lines indicate cleavage sites detected in wild-type plants (green) and fiery1 mutants (gray). Independent reads for each cut are shown as numbers next to the lines. A green box highlights a 15–17 bp dsRNA segment below the miRNA/miRNA* duplex.

    Techniques Used: Polymerase Chain Reaction, Amplification

    8) Product Images from "Isoforms S and L of MRPL33 from alternative splicing have isoform-specific roles in the chemoresponse to epirubicin in gastric cancer cells via the PI3K/AKT signaling pathway"

    Article Title: Isoforms S and L of MRPL33 from alternative splicing have isoform-specific roles in the chemoresponse to epirubicin in gastric cancer cells via the PI3K/AKT signaling pathway

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2019.4728

    Chemoresponse to epirubicin is regulated by MRPL33-L and MRPL33-S in gastric cancer. (A) Histogram of the chemoresponse of AGS cell groups (AGS control, plenti-vector, plenti-MRPL33-L and plenti-MRPL33-S), which were treated with epirubicin (0.003, 0.03, 0.3, 3 and 30 µ M) for 72 h. (B) Fluorescent staining of nuclei in AGS cell groups treated with 0.3 µ M epirubicin for 72 h. (C) Histogram of the chemoresponse of MGC-803 cell groups (MGC-803 control, plenti-vector, plenti-MRPL33-L and plenti-MRPL33-S), which were treated with epirubicin (0.003, 0.03, 0.3, 3 and 30 µ M) for 72 h. (D) Fluorescent staining of nuclei in MGC-803 cell groups treated with 0.3 µ M epirubicin for 72 h. Three independent biological replicates were performed and data were presented as the mean ± standard deviation. * P
    Figure Legend Snippet: Chemoresponse to epirubicin is regulated by MRPL33-L and MRPL33-S in gastric cancer. (A) Histogram of the chemoresponse of AGS cell groups (AGS control, plenti-vector, plenti-MRPL33-L and plenti-MRPL33-S), which were treated with epirubicin (0.003, 0.03, 0.3, 3 and 30 µ M) for 72 h. (B) Fluorescent staining of nuclei in AGS cell groups treated with 0.3 µ M epirubicin for 72 h. (C) Histogram of the chemoresponse of MGC-803 cell groups (MGC-803 control, plenti-vector, plenti-MRPL33-L and plenti-MRPL33-S), which were treated with epirubicin (0.003, 0.03, 0.3, 3 and 30 µ M) for 72 h. (D) Fluorescent staining of nuclei in MGC-803 cell groups treated with 0.3 µ M epirubicin for 72 h. Three independent biological replicates were performed and data were presented as the mean ± standard deviation. * P

    Techniques Used: Plasmid Preparation, Staining, Standard Deviation

    Effects of MRPL33-L and MRPL33-S overexpression in AGS gastric cancer cells. (A) Volcano plot and (B) heatmap indicating upregu-lated and downregulated genes in plenti-MRPL33-L-transfected cells. (C) Volcano plot and (D) heatmap indicating upregulated and downregulated genes in plenti-MRPL33-S-transfected cells. (E) Venn diagram and (F) heatmap showing the number of overlapping DEGs in plenti-MRPL33-L and plenti-MRPL33-S-transfected cells. (G) Gene Ontology analysis of DEGs in plenti-MRPL33-L and plenti-MRPL33-S-transfected cells. (H) KEGG pathway analysis of DEGs in plenti-MRPL33-L and plenti-MRPL33-S-transfected cells. MRPL33, mitochondrial ribosomal protein L33; L, long variant; S, short variant; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes.
    Figure Legend Snippet: Effects of MRPL33-L and MRPL33-S overexpression in AGS gastric cancer cells. (A) Volcano plot and (B) heatmap indicating upregu-lated and downregulated genes in plenti-MRPL33-L-transfected cells. (C) Volcano plot and (D) heatmap indicating upregulated and downregulated genes in plenti-MRPL33-S-transfected cells. (E) Venn diagram and (F) heatmap showing the number of overlapping DEGs in plenti-MRPL33-L and plenti-MRPL33-S-transfected cells. (G) Gene Ontology analysis of DEGs in plenti-MRPL33-L and plenti-MRPL33-S-transfected cells. (H) KEGG pathway analysis of DEGs in plenti-MRPL33-L and plenti-MRPL33-S-transfected cells. MRPL33, mitochondrial ribosomal protein L33; L, long variant; S, short variant; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes.

    Techniques Used: Over Expression, Transfection, Variant Assay

    Chemoresponse to epirubicin is dependent on the PI3K/AKT/CREB/apoptosis axis, which is regulated by MRPL33-L and MRPL33-S in gastric cancer cells. (A) Western blot analysis and (B) corresponding histogram of ratio of p-AKT/AKT, ratio of p-CREB/CREB, Mcl-1 and Bcl-2 expression levels in the AGS cell groups (control, plenti-vector-plentil-MRPL33-S and plenti-MRPL33-L-transfected), with or without 0.3 µ M epirubicin. (C) Western blot analysis and (D) corresponding histogram of ratio of p-AKT/AKT, ratio of p-CREB/CREB, Mcl-1 and Bcl-2 expression levels in the MGC-803 cell groups (control, plenti-vector, plentil-MRPL33-S and plenti-MRPL33-L), with or without 0.3 µ M epirubicin. (E) Histograms of chemoresponse in AGS cell groups and (F) MGC-803 cell groups treated with 0.3 µ M epirubicin for 72 h. Three independent biological replicates were performed and data were presented as the mean ± standard deviation. * P
    Figure Legend Snippet: Chemoresponse to epirubicin is dependent on the PI3K/AKT/CREB/apoptosis axis, which is regulated by MRPL33-L and MRPL33-S in gastric cancer cells. (A) Western blot analysis and (B) corresponding histogram of ratio of p-AKT/AKT, ratio of p-CREB/CREB, Mcl-1 and Bcl-2 expression levels in the AGS cell groups (control, plenti-vector-plentil-MRPL33-S and plenti-MRPL33-L-transfected), with or without 0.3 µ M epirubicin. (C) Western blot analysis and (D) corresponding histogram of ratio of p-AKT/AKT, ratio of p-CREB/CREB, Mcl-1 and Bcl-2 expression levels in the MGC-803 cell groups (control, plenti-vector, plentil-MRPL33-S and plenti-MRPL33-L), with or without 0.3 µ M epirubicin. (E) Histograms of chemoresponse in AGS cell groups and (F) MGC-803 cell groups treated with 0.3 µ M epirubicin for 72 h. Three independent biological replicates were performed and data were presented as the mean ± standard deviation. * P

    Techniques Used: Western Blot, Expressing, Plasmid Preparation, Transfection, Standard Deviation

    Expression of MRPL33-L and MRPL33-S in gastric cancer. (A) Schematic diagram of the splice variants of MRPL33, including or lacking alternative exon 3 (MRPL33-L and MRPL33-S; top). Different amino acid sequences are presented in red font for of MRPL33-L and in blue font for MRPL33-S (bottom). (B) Agarose gel electrophoresis photograph and (C) corresponding scatter diagrams of expression levels of MRPL33-L and MRPL33-S in 10 paired clinical specimens of tumor and matched adjacent normal tissues from patients with gastric cancer. (D) Agarose gel electrophoresis photograph and (E) corresponding histograms of expression levels of MRPL33-L and MRPL33-S in AGS and MGC-803 cells. Three independent biological replicates were performed and data were presented as the mean ± standard deviation. *** P
    Figure Legend Snippet: Expression of MRPL33-L and MRPL33-S in gastric cancer. (A) Schematic diagram of the splice variants of MRPL33, including or lacking alternative exon 3 (MRPL33-L and MRPL33-S; top). Different amino acid sequences are presented in red font for of MRPL33-L and in blue font for MRPL33-S (bottom). (B) Agarose gel electrophoresis photograph and (C) corresponding scatter diagrams of expression levels of MRPL33-L and MRPL33-S in 10 paired clinical specimens of tumor and matched adjacent normal tissues from patients with gastric cancer. (D) Agarose gel electrophoresis photograph and (E) corresponding histograms of expression levels of MRPL33-L and MRPL33-S in AGS and MGC-803 cells. Three independent biological replicates were performed and data were presented as the mean ± standard deviation. *** P

    Techniques Used: Expressing, Agarose Gel Electrophoresis, Standard Deviation

    Analysis of the PI3K/AKT signaling pathway based on the KEGG database. (A) PPI network analysis of 36 target genes involved in the PI3K/AKT signaling pathway in plenti-MRPL33-L-transfected cells and in (B) plenti-MRPL33-S-transfected cells. (C) Map of the PI3K/AKT signaling pathway with the 36 target genes based on the KEGG database. Red and blue represent the upregulation and downregulation of genes, respectively. The size of circle in (A) and (B) indicates significance based on P-value. The square and rounded square in (C) represent genes in plenti-MRPL33-L- and plenti-MRPL33-S-trans-fected cells, respectively. * P
    Figure Legend Snippet: Analysis of the PI3K/AKT signaling pathway based on the KEGG database. (A) PPI network analysis of 36 target genes involved in the PI3K/AKT signaling pathway in plenti-MRPL33-L-transfected cells and in (B) plenti-MRPL33-S-transfected cells. (C) Map of the PI3K/AKT signaling pathway with the 36 target genes based on the KEGG database. Red and blue represent the upregulation and downregulation of genes, respectively. The size of circle in (A) and (B) indicates significance based on P-value. The square and rounded square in (C) represent genes in plenti-MRPL33-L- and plenti-MRPL33-S-trans-fected cells, respectively. * P

    Techniques Used: Transfection

    9) Product Images from "Increased heme synthesis in yeast induces a metabolic switch from fermentation to respiration even under conditions of glucose repression"

    Article Title: Increased heme synthesis in yeast induces a metabolic switch from fermentation to respiration even under conditions of glucose repression

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.790923

    Overexpression of HEM3 or HEM12 increases cellular heme level and induces mitochondrial respiration. Cellular heme levels ( A ), oxygen consumption ( B ), cellular ATP levels ( C ), and relative mRNA levels of the indicated genes ( D ) were determined in wild-type cells containing either the control plasmid (TZ1099) or high-copy-number plasmid expressing HEM3 ( HEM3-oe ; strain TZ1100) or HEM12 ( HEM12-oe ; strain TZ1101). The cells were pregrown under selection in synthetic complete medium and inoculated to an A 600 nm of 0.2 into YEP medium containing 2% glucose and grown for two generations. The experiments were repeated three times, and the results are shown as means ± S.D. ( error bars ). Values that are statistically different ( p
    Figure Legend Snippet: Overexpression of HEM3 or HEM12 increases cellular heme level and induces mitochondrial respiration. Cellular heme levels ( A ), oxygen consumption ( B ), cellular ATP levels ( C ), and relative mRNA levels of the indicated genes ( D ) were determined in wild-type cells containing either the control plasmid (TZ1099) or high-copy-number plasmid expressing HEM3 ( HEM3-oe ; strain TZ1100) or HEM12 ( HEM12-oe ; strain TZ1101). The cells were pregrown under selection in synthetic complete medium and inoculated to an A 600 nm of 0.2 into YEP medium containing 2% glucose and grown for two generations. The experiments were repeated three times, and the results are shown as means ± S.D. ( error bars ). Values that are statistically different ( p

    Techniques Used: Over Expression, Plasmid Preparation, Expressing, Selection

    10) Product Images from "Protein tyrosine O-glycosylation—A rather unexplored prokaryotic glycosylation system"

    Article Title: Protein tyrosine O-glycosylation—A rather unexplored prokaryotic glycosylation system

    Journal: Glycobiology

    doi: 10.1093/glycob/cwq035

    RT-PCR analysis of total RNA of P. alvei CCM 2051 T . Reverse transcription was performed with the specific primer 4r targeted to wsfA (lanes 2–7) or 8r annealing to wsfF (lanes 9–20). Subsequent cDNA amplification was carried out with primer pairs 2f/2r annealing to galU / wzm (lanes 2–4), with 3f/3r targeted to wzt / wsfA (lanes 5–7), with 4f/4r annealing to wsfA (lanes 9–11), with 5f/5r targeted to wsfC / wsfD (lanes 12–14), with 6f/6r annealing to wsfD / wsfE (lanes 15–17) and with 8f/8r amplifying rmlB / wsfF (lanes 18–20). Lanes (a) show the specific PCR amplification products, using reverse transcribed single-strand cDNA as template; lanes (b) show control reactions, using DNase I-treated RNA without the cDNA-generating step as PCR template; lanes (c) show positive controls using genomic DNA as template. The 1-kb DNA Plus marker (Invitrogen) was used as DNA size marker (lanes 1, 8 and 20).
    Figure Legend Snippet: RT-PCR analysis of total RNA of P. alvei CCM 2051 T . Reverse transcription was performed with the specific primer 4r targeted to wsfA (lanes 2–7) or 8r annealing to wsfF (lanes 9–20). Subsequent cDNA amplification was carried out with primer pairs 2f/2r annealing to galU / wzm (lanes 2–4), with 3f/3r targeted to wzt / wsfA (lanes 5–7), with 4f/4r annealing to wsfA (lanes 9–11), with 5f/5r targeted to wsfC / wsfD (lanes 12–14), with 6f/6r annealing to wsfD / wsfE (lanes 15–17) and with 8f/8r amplifying rmlB / wsfF (lanes 18–20). Lanes (a) show the specific PCR amplification products, using reverse transcribed single-strand cDNA as template; lanes (b) show control reactions, using DNase I-treated RNA without the cDNA-generating step as PCR template; lanes (c) show positive controls using genomic DNA as template. The 1-kb DNA Plus marker (Invitrogen) was used as DNA size marker (lanes 1, 8 and 20).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Marker

    11) Product Images from "Cryptic Exon Incorporation Occurs in Alzheimer’s Brain lacking TDP-43 Inclusion but Exhibiting Nuclear Clearance of TDP-43"

    Article Title: Cryptic Exon Incorporation Occurs in Alzheimer’s Brain lacking TDP-43 Inclusion but Exhibiting Nuclear Clearance of TDP-43

    Journal: Acta neuropathologica

    doi: 10.1007/s00401-017-1701-2

    Further validation of cryptic exon incorporation in AD brain tissue. ( A ) Diagram of RT-PCR detection strategy to amplify across the cryptic exon splice junction of HDGFRP2 . ( B ) Sequencing confirmed 263 bp HDGFRP2 RT-PCR products were detected in the same cases that showed cryptic exon incorporation of GPSM2 and ATG4B . Similarly, GPSM2 and ATG4B negative cases did not display HDGFRP2 RT-PCR fragment (the band as outlined in case #4 was confirmed to be negative by sequencing). +: inclusions seen in both amygdala and dentate gyrus of hippocampus; ─*: inclusions only seen in amygdala; ─: no solid inclusions. C, control. HIP, hippocampus.
    Figure Legend Snippet: Further validation of cryptic exon incorporation in AD brain tissue. ( A ) Diagram of RT-PCR detection strategy to amplify across the cryptic exon splice junction of HDGFRP2 . ( B ) Sequencing confirmed 263 bp HDGFRP2 RT-PCR products were detected in the same cases that showed cryptic exon incorporation of GPSM2 and ATG4B . Similarly, GPSM2 and ATG4B negative cases did not display HDGFRP2 RT-PCR fragment (the band as outlined in case #4 was confirmed to be negative by sequencing). +: inclusions seen in both amygdala and dentate gyrus of hippocampus; ─*: inclusions only seen in amygdala; ─: no solid inclusions. C, control. HIP, hippocampus.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing

    12) Product Images from "Chronic Hyperinsulinemia Causes Selective Insulin Resistance and Down-regulates Uncoupling Protein 3 (UCP3) through the Activation of Sterol Regulatory Element-binding Protein (SREBP)-1 Transcription Factor in the Mouse Heart *"

    Article Title: Chronic Hyperinsulinemia Causes Selective Insulin Resistance and Down-regulates Uncoupling Protein 3 (UCP3) through the Activation of Sterol Regulatory Element-binding Protein (SREBP)-1 Transcription Factor in the Mouse Heart *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.673988

    Acute stimulation of insulin signaling in mouse heart inhibits UCP3 expression. A , time course analysis of insulin signaling activation in the heart of overnight fasted mice ( n = 37). Representative immunoblots for the phosphorylation of Akt at Thr-308 and Ser-473, the phosphorylation of the direct Akt target GSK-3β at Ser-9, and the phosphorylation of the p44/42 MAPK at both Thr-202 and Tyr-204 are shown. β-Tubulin levels are shown as a loading control. *, p
    Figure Legend Snippet: Acute stimulation of insulin signaling in mouse heart inhibits UCP3 expression. A , time course analysis of insulin signaling activation in the heart of overnight fasted mice ( n = 37). Representative immunoblots for the phosphorylation of Akt at Thr-308 and Ser-473, the phosphorylation of the direct Akt target GSK-3β at Ser-9, and the phosphorylation of the p44/42 MAPK at both Thr-202 and Tyr-204 are shown. β-Tubulin levels are shown as a loading control. *, p

    Techniques Used: Expressing, Activation Assay, Mouse Assay, Western Blot

    13) Product Images from "Novel Features of a PIWI-Like Protein Homolog in the Parasitic Protozoan Leishmania"

    Article Title: Novel Features of a PIWI-Like Protein Homolog in the Parasitic Protozoan Leishmania

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052612

    Genomic inactivation of the  PIWI  gene in  L. infantum  and  L. major  strains leads to a decrease in amastigote growth and disease pathology. (A, upper panel) Strategy to inactivate the  L. infantum PIWI  gene ( Lin PIWI (−/−) ) by genetic replacement. Both alleles of the  LinPIWI  single copy gene were replaced by the hygromycin phosphotransferase gene ( HYG ) by a loss of heterozygocity. (A, bottom panel) Southern blot hybridization of  L. infantum  genomic DNA digested with NcoI using the  PIWI  5′ flank sequence as a probe. In  Lin PIWI (+/+) , only a 2.3 kb band which corresponds to the wild type alleles was detected. In  Lin PIWI (+/−)  clones (C1, C2 and C3), in addition to the wild type allele, one more band of 1.0 kb (for the  HYG  gene integration) was detected. In  Lin PIWI (−/−) , only one band of 1.0 kb (for the  HYG  gene replacement) was detected but not the 2.3 kb band. (B) Growth curve of  L. infantum  axenic amastigotes for  Lin PIWI (+/+)  and  Lin PIWI (−/−)  independent clones 3, 5 and 6. The growth pattern of  L. infantum  WT and  Lin PIWI (−/−)  clones on days 1, 3, 5 and 7 was analyzed by one-way ANOVA followed by a Tukey’s post-test using GraphPad Prism (version 3.03) software. Significant differences between the various groups are indicated (*,  P
    Figure Legend Snippet: Genomic inactivation of the PIWI gene in L. infantum and L. major strains leads to a decrease in amastigote growth and disease pathology. (A, upper panel) Strategy to inactivate the L. infantum PIWI gene ( Lin PIWI (−/−) ) by genetic replacement. Both alleles of the LinPIWI single copy gene were replaced by the hygromycin phosphotransferase gene ( HYG ) by a loss of heterozygocity. (A, bottom panel) Southern blot hybridization of L. infantum genomic DNA digested with NcoI using the PIWI 5′ flank sequence as a probe. In Lin PIWI (+/+) , only a 2.3 kb band which corresponds to the wild type alleles was detected. In Lin PIWI (+/−) clones (C1, C2 and C3), in addition to the wild type allele, one more band of 1.0 kb (for the HYG gene integration) was detected. In Lin PIWI (−/−) , only one band of 1.0 kb (for the HYG gene replacement) was detected but not the 2.3 kb band. (B) Growth curve of L. infantum axenic amastigotes for Lin PIWI (+/+) and Lin PIWI (−/−) independent clones 3, 5 and 6. The growth pattern of L. infantum WT and Lin PIWI (−/−) clones on days 1, 3, 5 and 7 was analyzed by one-way ANOVA followed by a Tukey’s post-test using GraphPad Prism (version 3.03) software. Significant differences between the various groups are indicated (*, P

    Techniques Used: Southern Blot, Hybridization, Sequencing, Clone Assay, Software

    14) Product Images from "DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFR T790m Mutation"

    Article Title: DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFR T790m Mutation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067782

    Comparison of PNA-LNA sensitivity of detection for the T790M EGFR mutation. (A) Real-time PCR plot represents serially diluted PCR product with T790M mutation (1%, 0.1% and 0.01%) into amplified product from wild type DNA, by conventional PNA-LNA PCR. (A, right) Corresponding plot of the log concentration of T790M mutant DNA versus threshold cycle number. Graph shows mutant T790M serial dilution following conventional PNA-LNA PCR. (B) Real-time PCR plot shows an increased level of detection when PNA-LNA is applied to a sample that has undergone two rounds of mutant enrichment by DISSECT. (B, right) Corresponding graph shows mutant T790M serial dilution following PNA-LNA PCR after DISSECT.
    Figure Legend Snippet: Comparison of PNA-LNA sensitivity of detection for the T790M EGFR mutation. (A) Real-time PCR plot represents serially diluted PCR product with T790M mutation (1%, 0.1% and 0.01%) into amplified product from wild type DNA, by conventional PNA-LNA PCR. (A, right) Corresponding plot of the log concentration of T790M mutant DNA versus threshold cycle number. Graph shows mutant T790M serial dilution following conventional PNA-LNA PCR. (B) Real-time PCR plot shows an increased level of detection when PNA-LNA is applied to a sample that has undergone two rounds of mutant enrichment by DISSECT. (B, right) Corresponding graph shows mutant T790M serial dilution following PNA-LNA PCR after DISSECT.

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Concentration Assay, Serial Dilution

    T790M EGFR mutant enrichment analysis by HRM and Sanger Sequencing. (A) HRM analysis plots and Sanger Sequencing chromatograms of serial dilutions of T790M EGFR mutations versus wild type DNA following conventional PCR. (B) The detection after mutant enrichment by conventional PNA-LNA PCR using an initial mutant abundance of 1% and 0.1% T790M, c.2369C > T. (C) Improved detection after mutant enrichment from two rounds of DISSECT followed by conventional PNA-LNA PCR from an initial mutant abundance of 1% and 0.01% T790M, c.2369C > T.
    Figure Legend Snippet: T790M EGFR mutant enrichment analysis by HRM and Sanger Sequencing. (A) HRM analysis plots and Sanger Sequencing chromatograms of serial dilutions of T790M EGFR mutations versus wild type DNA following conventional PCR. (B) The detection after mutant enrichment by conventional PNA-LNA PCR using an initial mutant abundance of 1% and 0.1% T790M, c.2369C > T. (C) Improved detection after mutant enrichment from two rounds of DISSECT followed by conventional PNA-LNA PCR from an initial mutant abundance of 1% and 0.01% T790M, c.2369C > T.

    Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction

    15) Product Images from "Genetic Confirmation of the Role of Sulfopyruvate Decarboxylase in Coenzyme M Biosynthesis in Methanococcus maripaludis"

    Article Title: Genetic Confirmation of the Role of Sulfopyruvate Decarboxylase in Coenzyme M Biosynthesis in Methanococcus maripaludis

    Journal: Archaea

    doi: 10.1155/2013/185250

    Alignment of partial amino acid sequences of the enzyme sulfopyruvate decarboxylase from methanoarchaea, from top to bottom: Methanothermobacter thermautotrophicus, Methanospirillum hungatei, Methanosarcina acetivorans, Methanocaldococcus jannaschii , and Methanococcus maripaludis . The arrows signify insertion positions of the Tn5〈KAN-2-pac〉 transposon into the gene comE (558 bp) of M. maripaludis . The insertion at 336 bp (112 aa) corresponds to the mutation in strain M. maripaludis S201, and the insertion at 395 bp (131 aa) corresponds to the mutation in strain M. maripaludis S202. Blue boxes containing aligned amino acids correspond to the conserved thiamine pyrophosphate-binding domain. Asterisks, double dots, and single dots denote positions that contain fully conserved amino acid residues, groups of strongly similar residues, and groups of weakly similar residues, respectively.
    Figure Legend Snippet: Alignment of partial amino acid sequences of the enzyme sulfopyruvate decarboxylase from methanoarchaea, from top to bottom: Methanothermobacter thermautotrophicus, Methanospirillum hungatei, Methanosarcina acetivorans, Methanocaldococcus jannaschii , and Methanococcus maripaludis . The arrows signify insertion positions of the Tn5〈KAN-2-pac〉 transposon into the gene comE (558 bp) of M. maripaludis . The insertion at 336 bp (112 aa) corresponds to the mutation in strain M. maripaludis S201, and the insertion at 395 bp (131 aa) corresponds to the mutation in strain M. maripaludis S202. Blue boxes containing aligned amino acids correspond to the conserved thiamine pyrophosphate-binding domain. Asterisks, double dots, and single dots denote positions that contain fully conserved amino acid residues, groups of strongly similar residues, and groups of weakly similar residues, respectively.

    Techniques Used: Mutagenesis, Binding Assay

    16) Product Images from "Mismatch Repair Genes Mlh1 and Mlh3 Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches"

    Article Title: Mismatch Repair Genes Mlh1 and Mlh3 Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003930

    The 129 and B6 3′-flanking regions of  Mlh1  confer differential mRNA regulation. Investigation of the regulatory potential of B6 and 129 immediate (A) 5′- and (B) 3′-flanking regions of  Mlh1  using dual luciferase reporter assays. (A) The immediate 5′-flanking region of  Mlh1  containing 17 B6-129 polymorphisms (2,441 bp) was used to drive firefly luciferase expression. (B) The immediate 3′-flanking region of  Mlh1  ( i–iv ) containing either 19, 15, 4 or 1 B6-129 polymorphism(s) (1,676 bp, 1,280 bp, 591 bp and 205 bp, respectively) was cloned downstream of a firefly luciferase gene. “Swap” constructs ( v ) of the immediate 3′-flanking region of  Mlh1  containing either 4, 5 or 10 129 polymorphisms (530 bp, 438 bp and 708 bp, respectively; total 1676 bp) were cloned downstream of a firefly luciferase gene. Relative luciferase activity was determined by normalization to internal  Renilla  luminescence and determined relative to the analogous B6 construct. B6-129 polymorphisms are represented by open triangles. Bar graphs represent mean ±SD. *,  p
    Figure Legend Snippet: The 129 and B6 3′-flanking regions of Mlh1 confer differential mRNA regulation. Investigation of the regulatory potential of B6 and 129 immediate (A) 5′- and (B) 3′-flanking regions of Mlh1 using dual luciferase reporter assays. (A) The immediate 5′-flanking region of Mlh1 containing 17 B6-129 polymorphisms (2,441 bp) was used to drive firefly luciferase expression. (B) The immediate 3′-flanking region of Mlh1 ( i–iv ) containing either 19, 15, 4 or 1 B6-129 polymorphism(s) (1,676 bp, 1,280 bp, 591 bp and 205 bp, respectively) was cloned downstream of a firefly luciferase gene. “Swap” constructs ( v ) of the immediate 3′-flanking region of Mlh1 containing either 4, 5 or 10 129 polymorphisms (530 bp, 438 bp and 708 bp, respectively; total 1676 bp) were cloned downstream of a firefly luciferase gene. Relative luciferase activity was determined by normalization to internal Renilla luminescence and determined relative to the analogous B6 construct. B6-129 polymorphisms are represented by open triangles. Bar graphs represent mean ±SD. *, p

    Techniques Used: Luciferase, Expressing, Clone Assay, Construct, Activity Assay

    17) Product Images from "Genetic Variation of Human Papillomavirus Type 16 in Individual Clinical Specimens Revealed by Deep Sequencing"

    Article Title: Genetic Variation of Human Papillomavirus Type 16 in Individual Clinical Specimens Revealed by Deep Sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080583

    Amplification of full-length HPV16 genomes by full-circle PCR. (A) PCR was performed with PrimeSTAR ® GXL DNA polymerase and HPV16-specific primer-pairs as indicated. The amounts of HPV16/pUC19 used for the PCR template are also indicated above. The PCR products were analyzed by agarose gel electrophoresis. M, DNA size markers. (B) Scheme for full-circle PCR. PrimeSTAR ® GXL DNA polymerase generates short and long DNA products with primer-pair 1742F/1873R. (C) Full-circle PCR with DNA extracted from W12 cells, clone 20863 (high-copy HPV16 episomes) (lane 2) and clone 20850 (low-copy HPV16 episomes) (lane 3). M, DNA size marker (lanes 1) (D) Full-circle PCR using DNA isolated from 7 clinical specimens: 5 LSIL (lanes 2 to 6), and 2 ICC (lanes 7 and 8). M, DNA size marker (lanes 1).
    Figure Legend Snippet: Amplification of full-length HPV16 genomes by full-circle PCR. (A) PCR was performed with PrimeSTAR ® GXL DNA polymerase and HPV16-specific primer-pairs as indicated. The amounts of HPV16/pUC19 used for the PCR template are also indicated above. The PCR products were analyzed by agarose gel electrophoresis. M, DNA size markers. (B) Scheme for full-circle PCR. PrimeSTAR ® GXL DNA polymerase generates short and long DNA products with primer-pair 1742F/1873R. (C) Full-circle PCR with DNA extracted from W12 cells, clone 20863 (high-copy HPV16 episomes) (lane 2) and clone 20850 (low-copy HPV16 episomes) (lane 3). M, DNA size marker (lanes 1) (D) Full-circle PCR using DNA isolated from 7 clinical specimens: 5 LSIL (lanes 2 to 6), and 2 ICC (lanes 7 and 8). M, DNA size marker (lanes 1).

    Techniques Used: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Isolation, Immunocytochemistry

    18) Product Images from "Direct Competition between hnRNP C and U2AF65 Protects the Transcriptome from the Exonization of Alu Elements"

    Article Title: Direct Competition between hnRNP C and U2AF65 Protects the Transcriptome from the Exonization of Alu Elements

    Journal: Cell

    doi: 10.1016/j.cell.2012.12.023

    Point Mutations that Impair hnRNP C Binding Promote Inclusion of the Alu Exon in the CD55 Minigene (A) Schematic overview of the minigene including the Alu exon (gray square), intronic regions (black lines), and two flanking exons (white squares) from the CD55 gene. The original sequence (WT) as well as the mutated sequence surrounding the 3′ and 5′ splice sites (3 mut and 5 mut , respectively; splice sites marked by arrowheads) are depicted below. Introduced point mutations are highlighted in black. (B) RT-PCR monitoring inclusion or suppression of the Alu exon in the minigenes with wild-type (WT) or mutated sequences (3 mut , 5 mut ) in HNRNPC knockdown (KD1 and KD2) and control HeLa cells (Ctrl). The corresponding capillary electrophoresis data is given in a gel-like representation with Alu exon inclusion and suppression indicated schematically on the right. (C) Average Alu exon inclusion in percent from three replicate RT-PCR experiments. Lines indicate relevant comparisons with asterisks representing different levels of significance ( ∗ p value
    Figure Legend Snippet: Point Mutations that Impair hnRNP C Binding Promote Inclusion of the Alu Exon in the CD55 Minigene (A) Schematic overview of the minigene including the Alu exon (gray square), intronic regions (black lines), and two flanking exons (white squares) from the CD55 gene. The original sequence (WT) as well as the mutated sequence surrounding the 3′ and 5′ splice sites (3 mut and 5 mut , respectively; splice sites marked by arrowheads) are depicted below. Introduced point mutations are highlighted in black. (B) RT-PCR monitoring inclusion or suppression of the Alu exon in the minigenes with wild-type (WT) or mutated sequences (3 mut , 5 mut ) in HNRNPC knockdown (KD1 and KD2) and control HeLa cells (Ctrl). The corresponding capillary electrophoresis data is given in a gel-like representation with Alu exon inclusion and suppression indicated schematically on the right. (C) Average Alu exon inclusion in percent from three replicate RT-PCR experiments. Lines indicate relevant comparisons with asterisks representing different levels of significance ( ∗ p value

    Techniques Used: Binding Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Electrophoresis

    hnRNP C Suppresses the Exonization of an Alu Element in the NUP133 Gene through Competition with U2AF65, Related to Figure 5 (A) Genome browser view including an Alu element (orange) plus the downstream exon within the NUP133 gene on chromosome 1 (229,601,700-229,601,100). hnRNP C iCLIP data (blue) show crosslinking at the upstream and the linker U-tract of the Alu element (corresponding thymidines are highlighted in red at the bottom). Little U2AF65 crosslinking (purple) is detected at the U-tracts in control HeLa cells, while strong crosslinking is observed in the HNRNPC knockdowns (KD1 and KD2). RNA-seq data (green) show exonization of the Alu element in both knockdowns, giving rise to three different Alu exon variants. The corresponding isoforms are schematically indicated: Alu suppression in isoform S, Alu inclusion as a cassette exon in isoform 1 and 2 (differing in the usage of an upstream or downstream 5′ splice site, respectively) and Alu inclusion using an alternative 3‘ splice site for the downstream exon (isoform 3). The wild-type (WT) sequence surrounding the 3′ and 5′ splice sites (arrowheads) including the introduced point mutations (3 mut and 5 mut ) are shown below. The positions of the point mutations are highlighted in the sequences by black squares. (B) U-to-C transitions promote Alu exonization in the presence of hnRNP C. Shown is the gel-like view of capillary electrophoresis of RT-PCR analysis of minigenes containing the Alu element in the NUP133 gene described in (A) with two flanking exons. The wild-type (WT) minigene shows no Alu exonization in control HeLa cells (Ctrl) and a significant increase in isoforms 1-3 in the HNRNPC knockdown (KD1 and KD2). A mutant minigene containing three T-to-C transitions in the upstream U-tract (3 mut ) shows significant inclusion of isoforms 1-3 in control HeLa cells. Additional T-to-C transitions in the linker U-tract (5 mut ) further elevate the inclusion of isoforms 1-3 in control HeLa cells and prevent any further regulation by hnRNP C (KD1 and KD2). The sizes corresponding to the different analyzed isoforms are schematically indicated on the right. (C) Average Alu exon inclusion in percent for three replicate RT-PCR experiments as described in (B). The different isoforms are indicated by shades of gray (light gray: isoform 1; medium gray: isoform 2; dark gray: isoform 3). Lines indicate relevant comparisons with asterisks indicating different levels of significance for changes in the summed inclusion isoforms ( ∗ : p value
    Figure Legend Snippet: hnRNP C Suppresses the Exonization of an Alu Element in the NUP133 Gene through Competition with U2AF65, Related to Figure 5 (A) Genome browser view including an Alu element (orange) plus the downstream exon within the NUP133 gene on chromosome 1 (229,601,700-229,601,100). hnRNP C iCLIP data (blue) show crosslinking at the upstream and the linker U-tract of the Alu element (corresponding thymidines are highlighted in red at the bottom). Little U2AF65 crosslinking (purple) is detected at the U-tracts in control HeLa cells, while strong crosslinking is observed in the HNRNPC knockdowns (KD1 and KD2). RNA-seq data (green) show exonization of the Alu element in both knockdowns, giving rise to three different Alu exon variants. The corresponding isoforms are schematically indicated: Alu suppression in isoform S, Alu inclusion as a cassette exon in isoform 1 and 2 (differing in the usage of an upstream or downstream 5′ splice site, respectively) and Alu inclusion using an alternative 3‘ splice site for the downstream exon (isoform 3). The wild-type (WT) sequence surrounding the 3′ and 5′ splice sites (arrowheads) including the introduced point mutations (3 mut and 5 mut ) are shown below. The positions of the point mutations are highlighted in the sequences by black squares. (B) U-to-C transitions promote Alu exonization in the presence of hnRNP C. Shown is the gel-like view of capillary electrophoresis of RT-PCR analysis of minigenes containing the Alu element in the NUP133 gene described in (A) with two flanking exons. The wild-type (WT) minigene shows no Alu exonization in control HeLa cells (Ctrl) and a significant increase in isoforms 1-3 in the HNRNPC knockdown (KD1 and KD2). A mutant minigene containing three T-to-C transitions in the upstream U-tract (3 mut ) shows significant inclusion of isoforms 1-3 in control HeLa cells. Additional T-to-C transitions in the linker U-tract (5 mut ) further elevate the inclusion of isoforms 1-3 in control HeLa cells and prevent any further regulation by hnRNP C (KD1 and KD2). The sizes corresponding to the different analyzed isoforms are schematically indicated on the right. (C) Average Alu exon inclusion in percent for three replicate RT-PCR experiments as described in (B). The different isoforms are indicated by shades of gray (light gray: isoform 1; medium gray: isoform 2; dark gray: isoform 3). Lines indicate relevant comparisons with asterisks indicating different levels of significance for changes in the summed inclusion isoforms ( ∗ : p value

    Techniques Used: RNA Sequencing Assay, Sequencing, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Mutagenesis

    19) Product Images from "Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species"

    Article Title: Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species

    Journal: Biochemical Journal

    doi: 10.1042/BJ20131415

    Affinity-tag purification of BF-NanU and TF-NanU proteins ( A ) PCR-amplified C-terminally His 6 -tagged versions of BF-NanU ( BF1720 ) and TF-NanU ( TF0034 ) were expressed in E. coli BL21λ(DE3) cells containing relevant pET21a(+) derivatives and purified (see the Materials and methods section) before analysis by SDS/PAGE. Molecular mass is given on the left-hand side in kDa. ( B ) Purified BF-NanU (3 mg/ml), with and without pre-incubated Neu5Ac at an equimolar concentration, were sequentially applied to a HiLoad Superdex 200 PG gel-filtration column, calibrated with a gel-filtration standard of which the protein peaks and their corresponding elution volumes are represented by vertical lines. Both NanU and NanU–Neu5Ac migrated as globular species with an apparent molecular mass of ~58 kDa protein, approximating that of a monomer (57 kDa). mAU, milli-absorption unit.
    Figure Legend Snippet: Affinity-tag purification of BF-NanU and TF-NanU proteins ( A ) PCR-amplified C-terminally His 6 -tagged versions of BF-NanU ( BF1720 ) and TF-NanU ( TF0034 ) were expressed in E. coli BL21λ(DE3) cells containing relevant pET21a(+) derivatives and purified (see the Materials and methods section) before analysis by SDS/PAGE. Molecular mass is given on the left-hand side in kDa. ( B ) Purified BF-NanU (3 mg/ml), with and without pre-incubated Neu5Ac at an equimolar concentration, were sequentially applied to a HiLoad Superdex 200 PG gel-filtration column, calibrated with a gel-filtration standard of which the protein peaks and their corresponding elution volumes are represented by vertical lines. Both NanU and NanU–Neu5Ac migrated as globular species with an apparent molecular mass of ~58 kDa protein, approximating that of a monomer (57 kDa). mAU, milli-absorption unit.

    Techniques Used: Purification, Polymerase Chain Reaction, Amplification, SDS Page, Incubation, Concentration Assay, Filtration

    Sequence alignment of NanU with SusD family members The sequence alignment profile of the N-terminal region was generated using the Multalin Server ( http://multalin.toulouse.inra.fr/multalin ). Structural motifs predicted for BF1720 are presented on the top of the sequence alignment, where spirals represent α-helices. The kink in the α2 helix is indicated by an arrowhead. Conserved residues across the orthologues are highlighted in grey. The sources of these SusD proteins are as follows: BF1720 from B. fragilis NCTC 9343, BDI2495 from Parabacteroides distasonis A.T.C.C. 8503, TF0034 from T. forsythia A.T.C.C. 43037, BVU2431 from B. vulgatus A.T.C.C. 8482, and SusD from BT1043 from B. thetaiotaomicron VPI-5482 (PDB code 3CKC) and B. thetaiotaomicron VPI-5482 (PDB code 3EHN).
    Figure Legend Snippet: Sequence alignment of NanU with SusD family members The sequence alignment profile of the N-terminal region was generated using the Multalin Server ( http://multalin.toulouse.inra.fr/multalin ). Structural motifs predicted for BF1720 are presented on the top of the sequence alignment, where spirals represent α-helices. The kink in the α2 helix is indicated by an arrowhead. Conserved residues across the orthologues are highlighted in grey. The sources of these SusD proteins are as follows: BF1720 from B. fragilis NCTC 9343, BDI2495 from Parabacteroides distasonis A.T.C.C. 8503, TF0034 from T. forsythia A.T.C.C. 43037, BVU2431 from B. vulgatus A.T.C.C. 8482, and SusD from BT1043 from B. thetaiotaomicron VPI-5482 (PDB code 3CKC) and B. thetaiotaomicron VPI-5482 (PDB code 3EHN).

    Techniques Used: Sequencing, Generated

    NanU expression in B. fragilis in response to different carbon sources B. fragilis NCTC 9343 cells cultured overnight on FA agar were subcultured on FA agar (16 h) (lane 4) or minimal medium agar supplemented with 15 mM glucose (lane 1), Neu5Ac (lane 2) or glucose (16 h) then Neu5Ac (16 h) (lane 3). Normalized amounts of proteins were stained with Coomassie Blue or probed with rat BF-NanU antiserum or rabbit anti-( E. coli GroEL) as described above. Molecular mass is given on the left-hand side in kDa.
    Figure Legend Snippet: NanU expression in B. fragilis in response to different carbon sources B. fragilis NCTC 9343 cells cultured overnight on FA agar were subcultured on FA agar (16 h) (lane 4) or minimal medium agar supplemented with 15 mM glucose (lane 1), Neu5Ac (lane 2) or glucose (16 h) then Neu5Ac (16 h) (lane 3). Normalized amounts of proteins were stained with Coomassie Blue or probed with rat BF-NanU antiserum or rabbit anti-( E. coli GroEL) as described above. Molecular mass is given on the left-hand side in kDa.

    Techniques Used: Expressing, Cell Culture, Staining

    Cellular localization of NanU in B. fragilis ( A ) Cell fractions isolated from B. fragilis NCTC 9343 cells were obtained by differential detergent fractionation as described in the Materials and methods section. Protein (10 μg) from the cytoplasmic (C), inner membrane (IM), outer membrane (OM) and 20× concentrated secreted (S) fractions were loaded into each lane, resolved by SDS/PAGE (10% gel) and stained with Coomassie Blue. Parallel gels were blotted on to nitrocellulose membranes and probed with BF-NanU rat antiserum and rabbit anti-( E. coli GroEL) as described above. The blots were incubated with HRP-conjugated anti-(rat goat IgG) or anti-(rabbit goat IgG) and visualized. Molecular mass is given on the left-hand side in kDa. ( B ) Representative micrographs of immobilized intact B. fragilis cells, probed with BF-NanU rat antiserum (lower row) or just PBS (upper row) and incubated in the dark with goat anti-(rat IgG–Alexa Fluor™ 488) (A488) and counterstaining with DAPI. Fluorescence was visualized at ×100 magnification using appropriate filters, with NanU (left-hand panels) and DNA (middle panels) shown in green and blue respectively, alongside a combined image (right-hand panels).
    Figure Legend Snippet: Cellular localization of NanU in B. fragilis ( A ) Cell fractions isolated from B. fragilis NCTC 9343 cells were obtained by differential detergent fractionation as described in the Materials and methods section. Protein (10 μg) from the cytoplasmic (C), inner membrane (IM), outer membrane (OM) and 20× concentrated secreted (S) fractions were loaded into each lane, resolved by SDS/PAGE (10% gel) and stained with Coomassie Blue. Parallel gels were blotted on to nitrocellulose membranes and probed with BF-NanU rat antiserum and rabbit anti-( E. coli GroEL) as described above. The blots were incubated with HRP-conjugated anti-(rat goat IgG) or anti-(rabbit goat IgG) and visualized. Molecular mass is given on the left-hand side in kDa. ( B ) Representative micrographs of immobilized intact B. fragilis cells, probed with BF-NanU rat antiserum (lower row) or just PBS (upper row) and incubated in the dark with goat anti-(rat IgG–Alexa Fluor™ 488) (A488) and counterstaining with DAPI. Fluorescence was visualized at ×100 magnification using appropriate filters, with NanU (left-hand panels) and DNA (middle panels) shown in green and blue respectively, alongside a combined image (right-hand panels).

    Techniques Used: Isolation, Fractionation, SDS Page, Staining, Incubation, Fluorescence

    Ability of nanOU genes to support growth on sialic acid ( A ) Growth kinetics of E. coli MG1655 Δ nanCnanR(amber) Δ ompR :: Tn10 ( tet ) (Δ nan ) mutant strains complemented in trans with the nanOU genes from T. forsythia (TF- nanOU , ○), B. fragilis (BF- nanOU , ∆) or the individual B. fragilis nanO (BF- nanO , ◇) or nanU (BF-nanU, ▼) expressed from pBAD18 (also included as a negative control, □) were monitored ( A 600 ) during culture at 37°C in M9 minimal media with 15 mM Neu5Ac. Results are means±S.D. from three separate biological replicates. ( B ) The Δ nan strain complemented with either B. fragilis nanO (BF- nanO , open symbols) or nanOU (BF- nanOU , close symbols) was incubated as above but with 6 (● and ○), 3 (◆ and ◇), 1 (■ and □) or 0.5 (▲ and ∆) mM Neu5Ac. Results are means±S.D. from three separate biological replicates. ( C and D ) The sialic acid transport and tonB -deletion mutant strains [Δ nanCnanR ( amber ) Δ ompR :: Tn10 ( tet ) Δ tonB :: FRT-Km-FRT –Δ nan Δ ton ] were complemented as in ( A ) and ( B ) in M9 medium with either 15 mM glucose ( C ) or Neu5Ac ( D ) as indicated and growth followed over time. Results are means±S.D.
    Figure Legend Snippet: Ability of nanOU genes to support growth on sialic acid ( A ) Growth kinetics of E. coli MG1655 Δ nanCnanR(amber) Δ ompR :: Tn10 ( tet ) (Δ nan ) mutant strains complemented in trans with the nanOU genes from T. forsythia (TF- nanOU , ○), B. fragilis (BF- nanOU , ∆) or the individual B. fragilis nanO (BF- nanO , ◇) or nanU (BF-nanU, ▼) expressed from pBAD18 (also included as a negative control, □) were monitored ( A 600 ) during culture at 37°C in M9 minimal media with 15 mM Neu5Ac. Results are means±S.D. from three separate biological replicates. ( B ) The Δ nan strain complemented with either B. fragilis nanO (BF- nanO , open symbols) or nanOU (BF- nanOU , close symbols) was incubated as above but with 6 (● and ○), 3 (◆ and ◇), 1 (■ and □) or 0.5 (▲ and ∆) mM Neu5Ac. Results are means±S.D. from three separate biological replicates. ( C and D ) The sialic acid transport and tonB -deletion mutant strains [Δ nanCnanR ( amber ) Δ ompR :: Tn10 ( tet ) Δ tonB :: FRT-Km-FRT –Δ nan Δ ton ] were complemented as in ( A ) and ( B ) in M9 medium with either 15 mM glucose ( C ) or Neu5Ac ( D ) as indicated and growth followed over time. Results are means±S.D.

    Techniques Used: Mutagenesis, Negative Control, Incubation

    20) Product Images from "High-Efficiency Targeted Editing of Large Viral Genomes by RNA-Guided Nucleases"

    Article Title: High-Efficiency Targeted Editing of Large Viral Genomes by RNA-Guided Nucleases

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004090

    Herpes simplex viral genome editing targeted by the CRISPR-Cas system. (A) Schematic of the HSV1 genome and the gRNA-206 target site in the UL23 gene. PAM sequences are highlighted in orange. The BsiWI restriction site used for RFLP is highlighted in aqua green. (B) The fragment amplified from an HSV1 genome using oligo 8 and oligo 9 was used in the RFLP assay. Co-expression of Cas9 and gRNA-206 in HSV1-infected cells leads to indel mutations in the TK gene of the HSV1 genome, which destroys the BsiWI restriction site. Arrows indicate fragments generated by BsiWI digestion. (C) Titration of HSV1 using an endpoint dilution assay in the presence or absence of 100 µg/ml ACV. The titers of total HSV1 progeny are represented by a blue bar. The titers of ACV-resistant HSV1 progeny virus are represented by an orange bar. N = 3; error bars show the means ± SEM. ND, not detected. (D) Sequences of indel mutations identified from eight ACV-resistant HSV1 strains. Red dashes, deletions; red bases, insertions. The incidence of each genotype is listed in the right-most column. (E) Strategy of Cas9:gRNA-induced HDR used to insert the EGFP gene into the HSV1 genome using donor DNA. (F) Titration of recombinant HSV1 using a plaque assay. HDR was induced by the Cas9 protein or Cas9:gRNA206. Blue bars represent the total number of macroscopically visible plaques; and green bars represent the number of green fluorescent plaques that were counted using a fluorescence microscope. N > 5; error bars show the means ± SEM. ND, not detected. (G) An isolated green HSV1 plaque that was observed using a fluorescence microscope and merged with an image of DAPI staining. Scale bar, 50 µm. (H) PCR amplification of HSV1 genomes extracted from a green fluorescent plaque using oligo 8 and oligo 9 to verify the lack of wild-type HSV1 contamination.
    Figure Legend Snippet: Herpes simplex viral genome editing targeted by the CRISPR-Cas system. (A) Schematic of the HSV1 genome and the gRNA-206 target site in the UL23 gene. PAM sequences are highlighted in orange. The BsiWI restriction site used for RFLP is highlighted in aqua green. (B) The fragment amplified from an HSV1 genome using oligo 8 and oligo 9 was used in the RFLP assay. Co-expression of Cas9 and gRNA-206 in HSV1-infected cells leads to indel mutations in the TK gene of the HSV1 genome, which destroys the BsiWI restriction site. Arrows indicate fragments generated by BsiWI digestion. (C) Titration of HSV1 using an endpoint dilution assay in the presence or absence of 100 µg/ml ACV. The titers of total HSV1 progeny are represented by a blue bar. The titers of ACV-resistant HSV1 progeny virus are represented by an orange bar. N = 3; error bars show the means ± SEM. ND, not detected. (D) Sequences of indel mutations identified from eight ACV-resistant HSV1 strains. Red dashes, deletions; red bases, insertions. The incidence of each genotype is listed in the right-most column. (E) Strategy of Cas9:gRNA-induced HDR used to insert the EGFP gene into the HSV1 genome using donor DNA. (F) Titration of recombinant HSV1 using a plaque assay. HDR was induced by the Cas9 protein or Cas9:gRNA206. Blue bars represent the total number of macroscopically visible plaques; and green bars represent the number of green fluorescent plaques that were counted using a fluorescence microscope. N > 5; error bars show the means ± SEM. ND, not detected. (G) An isolated green HSV1 plaque that was observed using a fluorescence microscope and merged with an image of DAPI staining. Scale bar, 50 µm. (H) PCR amplification of HSV1 genomes extracted from a green fluorescent plaque using oligo 8 and oligo 9 to verify the lack of wild-type HSV1 contamination.

    Techniques Used: CRISPR, Amplification, RFLP Assay, Expressing, Infection, Generated, Titration, Endpoint Dilution Assay, Recombinant, Plaque Assay, Fluorescence, Microscopy, Isolation, Staining, Polymerase Chain Reaction

    21) Product Images from "Genome-wide analysis of the RpoN regulon in Geobacter sulfurreducens"

    Article Title: Genome-wide analysis of the RpoN regulon in Geobacter sulfurreducens

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-10-331

    RpoN expression . (a) RpoN expression under different growth conditions. 1: NBAF; 2: NBH 2 F; 3: NBLF; 4: ammonium-free NBAF; 5: FWAFC; 6: FWH 2 FC; 7: FWLFC; 8: FWAF; 9: FWH 2 F; 10: FWLF. Media abbreviations were detailed in Methods. (b) RpoN over-expression. Total protein (5 μg) was separated by 10% SDS-PAGE and analyzed by Western blot analysis with the RpoN-specific antiserum. Two biological samples were shown for IPTG-induced WT V and RpoN + strains. IPTG was added at final concentration 1 mM.
    Figure Legend Snippet: RpoN expression . (a) RpoN expression under different growth conditions. 1: NBAF; 2: NBH 2 F; 3: NBLF; 4: ammonium-free NBAF; 5: FWAFC; 6: FWH 2 FC; 7: FWLFC; 8: FWAF; 9: FWH 2 F; 10: FWLF. Media abbreviations were detailed in Methods. (b) RpoN over-expression. Total protein (5 μg) was separated by 10% SDS-PAGE and analyzed by Western blot analysis with the RpoN-specific antiserum. Two biological samples were shown for IPTG-induced WT V and RpoN + strains. IPTG was added at final concentration 1 mM.

    Techniques Used: Expressing, Over Expression, SDS Page, Western Blot, Concentration Assay

    The rpoN gene cluster and the mutation schemes . (a) Genes surrounding rpoN are shown as open arrows. HP: conserved hypothetical protein with unknown function; YhbG: ABC transporter, ATP binding protein; YfiA: ribosomal subunit interface-associated sigma-54 modulation protein; HprK: Hpr(Ser) kinase/phosphorylase. Insertion of a kanamycin resistance cassette upstream or downstream of the intergenic region of the rpoN gene resulted in viable mutants (a). (b) Scheme showing attempts of construction of deletion of (i) the 5'-end, (ii) the whole, or (iii) the 3'-end of the rpoN coding region. (c) An extra copy of the rpoN gene was inserted on the chromosome and was under the control of the chloramphenicol resistance cassette promoter. (d) An extra copy of the rpoN gene was introduced in trans under the control of a lac promoter (constitutively expressed) or a taclac promoter (IPTG-inducible). The position of insertion of the antibiotic resistance cassette (kanamycin, Kan or gentamycin, Gm) is indicated with an inverted triangle and a vertical bar. The regions which were attempted to replace with the antibiotic resistance cassette insertion are indicated by dashed line.
    Figure Legend Snippet: The rpoN gene cluster and the mutation schemes . (a) Genes surrounding rpoN are shown as open arrows. HP: conserved hypothetical protein with unknown function; YhbG: ABC transporter, ATP binding protein; YfiA: ribosomal subunit interface-associated sigma-54 modulation protein; HprK: Hpr(Ser) kinase/phosphorylase. Insertion of a kanamycin resistance cassette upstream or downstream of the intergenic region of the rpoN gene resulted in viable mutants (a). (b) Scheme showing attempts of construction of deletion of (i) the 5'-end, (ii) the whole, or (iii) the 3'-end of the rpoN coding region. (c) An extra copy of the rpoN gene was inserted on the chromosome and was under the control of the chloramphenicol resistance cassette promoter. (d) An extra copy of the rpoN gene was introduced in trans under the control of a lac promoter (constitutively expressed) or a taclac promoter (IPTG-inducible). The position of insertion of the antibiotic resistance cassette (kanamycin, Kan or gentamycin, Gm) is indicated with an inverted triangle and a vertical bar. The regions which were attempted to replace with the antibiotic resistance cassette insertion are indicated by dashed line.

    Techniques Used: Mutagenesis, Binding Assay

    Characterization of the RpoN over-expression strain . Cell growth with fumarate as an electron acceptor was monitored by absorbance at 600 nm (a)(b). (a) acetate as the electron donor and fumarate as the electron acceptor (NBAF medium); (b): ammonia-free NBAF. Growth with Fe(III) as an electron acceptor was monitored by Fe(II) production (c) as well as cell numbers (d). Filled square: the WT V strain without IPTG; Empty square: the WT V strain with IPTG. Filled circle: the RpoN + strain without IPTG; empty circle: the RpoN + strain with IPTG. (a)-(d): Data are means ± standard deviations of triplicates. The production of pili was measured by agglutination assays (e). Data are means ± standard deviation of triplicates from two independent experiments (e).
    Figure Legend Snippet: Characterization of the RpoN over-expression strain . Cell growth with fumarate as an electron acceptor was monitored by absorbance at 600 nm (a)(b). (a) acetate as the electron donor and fumarate as the electron acceptor (NBAF medium); (b): ammonia-free NBAF. Growth with Fe(III) as an electron acceptor was monitored by Fe(II) production (c) as well as cell numbers (d). Filled square: the WT V strain without IPTG; Empty square: the WT V strain with IPTG. Filled circle: the RpoN + strain without IPTG; empty circle: the RpoN + strain with IPTG. (a)-(d): Data are means ± standard deviations of triplicates. The production of pili was measured by agglutination assays (e). Data are means ± standard deviation of triplicates from two independent experiments (e).

    Techniques Used: Over Expression, Agglutination, Standard Deviation

    22) Product Images from "Direct detection of DNA methylation during single-molecule, real-time sequencing"

    Article Title: Direct detection of DNA methylation during single-molecule, real-time sequencing

    Journal: Nature methods

    doi: 10.1038/nmeth.1459

    Comparison of SMRT sequencing kinetics for DNA samples propagated within dam + E. coli and for the same samples after whole-genome amplification (WGA). The sample comprises a 3.7-kb subregion of a C. elegans fosmid cloned into an E. coli vector. ( a ) 50-bp window GC-content of the sample, plotted versus template position. ( b ) Average IPD at each template position within the dam + sample. ( c ) Average IPD at each template position within the WGA sample. ( d ) Ratio of the average IPDs ( dam + in (b) divided by WGA in (c)), plotted versus template position. Positions with a GATC context, where methylation of adenine at the sequence motif GATC is expected, are denoted by black squares, and all other positions are denoted by open blue circles. Error bars at the GATC positions denote the s.e.m. IPD ratio at those positions (average n = 106 measurements at each position). For comparison, the mean ± s.d. of all IPD ratios at non-GATC positions (open blue circles) is 1.00 ± 0.24 ( n is ∼389,000 measurements). Average sequencing coverage across this fosmid region was 121-fold for the dam + sample and 91-fold for the WGA sample.
    Figure Legend Snippet: Comparison of SMRT sequencing kinetics for DNA samples propagated within dam + E. coli and for the same samples after whole-genome amplification (WGA). The sample comprises a 3.7-kb subregion of a C. elegans fosmid cloned into an E. coli vector. ( a ) 50-bp window GC-content of the sample, plotted versus template position. ( b ) Average IPD at each template position within the dam + sample. ( c ) Average IPD at each template position within the WGA sample. ( d ) Ratio of the average IPDs ( dam + in (b) divided by WGA in (c)), plotted versus template position. Positions with a GATC context, where methylation of adenine at the sequence motif GATC is expected, are denoted by black squares, and all other positions are denoted by open blue circles. Error bars at the GATC positions denote the s.e.m. IPD ratio at those positions (average n = 106 measurements at each position). For comparison, the mean ± s.d. of all IPD ratios at non-GATC positions (open blue circles) is 1.00 ± 0.24 ( n is ∼389,000 measurements). Average sequencing coverage across this fosmid region was 121-fold for the dam + sample and 91-fold for the WGA sample.

    Techniques Used: Sequencing, Whole Genome Amplification, Clone Assay, Plasmid Preparation, Methylation

    23) Product Images from "A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers"

    Article Title: A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

    Journal: Scientific Reports

    doi: 10.1038/srep11039

    Detection of MERS-CoV RNA from tissue culture-derived virions through reverse transcription and LAMP amplification (RT-LAMP). ( A ) Agarose gel electrophoretic characterization of RT-LAMP amplicons from samples containing RNA extracted from 60 PFU of MERS-CoV virons (or 2 μL 3E4 PFU/mL) (P-3) or samples lacking MERS-CoV RNA (N-1 and N-2). For each sample, the LAMP reaction was carried out at 55 o C for 1.5 hours and developed on a 1% agarose gel stained with ethidium bromide. ( B ) Glucometer responses for amplification reactions with RNAs extracted from 0.06 (P-1), 0.6 (P-2), 60 (P-3 and P-4) PFU MERS-CoV virions (2 μL) and with RNA-negative samples (N-1, N-2, and N-3). Samples underwent 1.5 hour or 10 min 55 o C RT-LAMP reactions, 1 hour 25 o C OSD, and 23 min 55 o C glucose generation with thermostable TmINV. The signals shown in N-1, N-2 and P-3 in Fig 3B were obtained from the same RT-LAMP reactions as the N-1, N-2, and P-3 samples shown in Fig 3A . It should be noted that the LAMP reaction for sample N-2 produces non-specific amplicons but these do not produce a false positive glucometer signal. The error bars represent standard deviations calculated from three parallel assays.
    Figure Legend Snippet: Detection of MERS-CoV RNA from tissue culture-derived virions through reverse transcription and LAMP amplification (RT-LAMP). ( A ) Agarose gel electrophoretic characterization of RT-LAMP amplicons from samples containing RNA extracted from 60 PFU of MERS-CoV virons (or 2 μL 3E4 PFU/mL) (P-3) or samples lacking MERS-CoV RNA (N-1 and N-2). For each sample, the LAMP reaction was carried out at 55 o C for 1.5 hours and developed on a 1% agarose gel stained with ethidium bromide. ( B ) Glucometer responses for amplification reactions with RNAs extracted from 0.06 (P-1), 0.6 (P-2), 60 (P-3 and P-4) PFU MERS-CoV virions (2 μL) and with RNA-negative samples (N-1, N-2, and N-3). Samples underwent 1.5 hour or 10 min 55 o C RT-LAMP reactions, 1 hour 25 o C OSD, and 23 min 55 o C glucose generation with thermostable TmINV. The signals shown in N-1, N-2 and P-3 in Fig 3B were obtained from the same RT-LAMP reactions as the N-1, N-2, and P-3 samples shown in Fig 3A . It should be noted that the LAMP reaction for sample N-2 produces non-specific amplicons but these do not produce a false positive glucometer signal. The error bars represent standard deviations calculated from three parallel assays.

    Techniques Used: Derivative Assay, Amplification, Agarose Gel Electrophoresis, Staining

    Sensitive and specific detection of synthetic MERS-CoV DNA (sORF1A) using LAMP-to-glucose transduction. The bar-graph presents glucometer responses to both non-target (RPOB) and different amounts of target (MERS-CoV sORF1A = MERS) templates. N-1, N-2, P-1 to P-4 were amplification reactions with the ORF1A.55 MERS-CoV primer set. Cognate targets (P) respond while non-cognate targets or negative controls (N) do not. Assays included a 1.5 hour 55 o C LAMP reaction, 1 hour 25 o C OSD, and 40 min 55 o C glucose generation using a commercial yeast invertase (yeast INV). The error bars represent standard deviations calculated from three parallel assays.
    Figure Legend Snippet: Sensitive and specific detection of synthetic MERS-CoV DNA (sORF1A) using LAMP-to-glucose transduction. The bar-graph presents glucometer responses to both non-target (RPOB) and different amounts of target (MERS-CoV sORF1A = MERS) templates. N-1, N-2, P-1 to P-4 were amplification reactions with the ORF1A.55 MERS-CoV primer set. Cognate targets (P) respond while non-cognate targets or negative controls (N) do not. Assays included a 1.5 hour 55 o C LAMP reaction, 1 hour 25 o C OSD, and 40 min 55 o C glucose generation using a commercial yeast invertase (yeast INV). The error bars represent standard deviations calculated from three parallel assays.

    Techniques Used: Transduction, Amplification

    Detection of synthetic ZEBOV DNA (ZEBOV-VP30) using LAMP-to-glucose transduction. Glucometer responses to 1.5E2 copies (P-1), 1.5E3 copies (P-2) and 1.5E4 copies (P-3) of synthetic ZEBOV VP30 (= ZEBOV) or to a DNA buffer control (0 copies, N-1). N-2 is the negative control for OSD signal transduction reaction and shows no response even in the presence of an amplification reaction seeded with 2E4 copies of the non-cognate MERS-CoV sORF1A (= MERS) template and appropriate MERS-specific primers. Reaction conditions were as in Fig 3 . The error bars represent standard deviations calculated from two parallel assays. The thermostable TmINV was used in these experiments.
    Figure Legend Snippet: Detection of synthetic ZEBOV DNA (ZEBOV-VP30) using LAMP-to-glucose transduction. Glucometer responses to 1.5E2 copies (P-1), 1.5E3 copies (P-2) and 1.5E4 copies (P-3) of synthetic ZEBOV VP30 (= ZEBOV) or to a DNA buffer control (0 copies, N-1). N-2 is the negative control for OSD signal transduction reaction and shows no response even in the presence of an amplification reaction seeded with 2E4 copies of the non-cognate MERS-CoV sORF1A (= MERS) template and appropriate MERS-specific primers. Reaction conditions were as in Fig 3 . The error bars represent standard deviations calculated from two parallel assays. The thermostable TmINV was used in these experiments.

    Techniques Used: Transduction, Negative Control, Amplification

    Stability and reproducibility of modified magnetic beads. ( A ) LAMP-to-glucose assays with 2E4 copies of MERS-CoV sORF1A target DNA (Day 1, Day 2, and Day 3, red) and buffer controls (Day 1, Day 2, and Day 3, green), carried out with conjugated magnetic beads (Inv-FPc/FP/MBs) that were prepared on three different days. On each day, three parallel assays were carried out with both the target MERS-CoV sORF1A DNA and buffer controls. A total of nine assays were performed for both the sORF1A target and buffer controls. Assay conditions were otherwise the same as in Fig 3 . ( B ) Time dependence of glucometer responses to LAMP amplicons generated from 2E4 copies of sORF1A using a single preparation of Inv-FPc/FP/MBs. The error bars represent standard deviations calculated from three parallel assays. The commercial yeast INV was used in these experiments.
    Figure Legend Snippet: Stability and reproducibility of modified magnetic beads. ( A ) LAMP-to-glucose assays with 2E4 copies of MERS-CoV sORF1A target DNA (Day 1, Day 2, and Day 3, red) and buffer controls (Day 1, Day 2, and Day 3, green), carried out with conjugated magnetic beads (Inv-FPc/FP/MBs) that were prepared on three different days. On each day, three parallel assays were carried out with both the target MERS-CoV sORF1A DNA and buffer controls. A total of nine assays were performed for both the sORF1A target and buffer controls. Assay conditions were otherwise the same as in Fig 3 . ( B ) Time dependence of glucometer responses to LAMP amplicons generated from 2E4 copies of sORF1A using a single preparation of Inv-FPc/FP/MBs. The error bars represent standard deviations calculated from three parallel assays. The commercial yeast INV was used in these experiments.

    Techniques Used: Modification, Magnetic Beads, Generated

    Using an OR gate to improve the robustness of sensing. ( A ) OR gate design. Either the ORF1A region or the upE region of amplicons produced from a MERS-CoV template could initiate OSD. The respective amplicons will displace either an Inv-ORF1A-Fc or an Inv-upE-FPc specific reporter strand bound to the complementary OR-P capture strand attached to magnetic beads. Either or both released invertases will lead to a glucometer response. ( B ) Performance of the OR gate with RNA extracted from 60 PFU MERS-CoV virons amplified with either no primers, with the upE.9 and ORF1A.55 individually, or with a combination of the upE.9 and ORF1A.55 primers. Reaction conditions were as in Fig 3 . The error bars represent standard deviations calculated from two parallel assays. The thermostable TmINV was used in these experiments.
    Figure Legend Snippet: Using an OR gate to improve the robustness of sensing. ( A ) OR gate design. Either the ORF1A region or the upE region of amplicons produced from a MERS-CoV template could initiate OSD. The respective amplicons will displace either an Inv-ORF1A-Fc or an Inv-upE-FPc specific reporter strand bound to the complementary OR-P capture strand attached to magnetic beads. Either or both released invertases will lead to a glucometer response. ( B ) Performance of the OR gate with RNA extracted from 60 PFU MERS-CoV virons amplified with either no primers, with the upE.9 and ORF1A.55 individually, or with a combination of the upE.9 and ORF1A.55 primers. Reaction conditions were as in Fig 3 . The error bars represent standard deviations calculated from two parallel assays. The thermostable TmINV was used in these experiments.

    Techniques Used: Produced, Magnetic Beads, Amplification

    24) Product Images from "The Atypical Occurrence of Two Biotin Protein Ligases in Francisella novicida Is Due to Distinct Roles in Virulence and Biotin Metabolism"

    Article Title: The Atypical Occurrence of Two Biotin Protein Ligases in Francisella novicida Is Due to Distinct Roles in Virulence and Biotin Metabolism

    Journal: mBio

    doi: 10.1128/mBio.00591-15

    F. novicida BplA is required for replication in mice. Mice were infected subcutaneously with 1 × 10 5 CFU of wild-type F. novicida U112 (WT) or the Δ bplA or Δ birA strain. At 48 h postinfection, skin samples obtained at the site of infection as well as the spleen and liver were harvested, and CFU were enumerated after plating. ***, P
    Figure Legend Snippet: F. novicida BplA is required for replication in mice. Mice were infected subcutaneously with 1 × 10 5 CFU of wild-type F. novicida U112 (WT) or the Δ bplA or Δ birA strain. At 48 h postinfection, skin samples obtained at the site of infection as well as the spleen and liver were harvested, and CFU were enumerated after plating. ***, P

    Techniques Used: Mouse Assay, Infection

    Sequence alignments of FTN_0568 (BplA, in blue) and FTN_0811 (BirA, in red) with E. coli BirA, the DNA binding domain of which is underlined in blue. The E. coli BirA structural elements are depicted above the alignments.
    Figure Legend Snippet: Sequence alignments of FTN_0568 (BplA, in blue) and FTN_0811 (BirA, in red) with E. coli BirA, the DNA binding domain of which is underlined in blue. The E. coli BirA structural elements are depicted above the alignments.

    Techniques Used: Sequencing, Binding Assay

    Growth of the derivatives of E. coli strain BM4062 carrying plasmid pBAD322 (empty vector) or pBAD322 derivatives encoding either the F. novicida BplA or BirA ligase. (A) Ligase expression was induced with arabinose; the strain BM4062 derivative with a wild-type arabinose operon was used to avoid arabinose toxicity. The biotin concentrations are shown below each plate. As shown at the higher biotin concentrations in the sectors containing the vector strain, the BirA encoded by the host strain mutant has weak ligase activity ( 21 ). (B) The strains were grown in the absence of arabinose induction and in the presence of glucose to repress the basal level of expression from the p araBAD promoter. The original strain BM4062 was used. Strain BM4062 containing the plasmid encoding BplA was streaked in the left-hand sectors, whereas the right-hand sectors contained the BirA-encoding plasmid. The plates were minimal medium M9 supplemented with 0.1% Casamino Acids, 40 µg/ml X-Gal, and 100 µg/ml ampicillin with 0.2% arabinose supplementation (A) and 0.4% glucose supplementation (B). The plates were incubated overnight at either 42°C (A) or 37°C (B).
    Figure Legend Snippet: Growth of the derivatives of E. coli strain BM4062 carrying plasmid pBAD322 (empty vector) or pBAD322 derivatives encoding either the F. novicida BplA or BirA ligase. (A) Ligase expression was induced with arabinose; the strain BM4062 derivative with a wild-type arabinose operon was used to avoid arabinose toxicity. The biotin concentrations are shown below each plate. As shown at the higher biotin concentrations in the sectors containing the vector strain, the BirA encoded by the host strain mutant has weak ligase activity ( 21 ). (B) The strains were grown in the absence of arabinose induction and in the presence of glucose to repress the basal level of expression from the p araBAD promoter. The original strain BM4062 was used. Strain BM4062 containing the plasmid encoding BplA was streaked in the left-hand sectors, whereas the right-hand sectors contained the BirA-encoding plasmid. The plates were minimal medium M9 supplemented with 0.1% Casamino Acids, 40 µg/ml X-Gal, and 100 µg/ml ampicillin with 0.2% arabinose supplementation (A) and 0.4% glucose supplementation (B). The plates were incubated overnight at either 42°C (A) or 37°C (B).

    Techniques Used: Plasmid Preparation, Expressing, Mutagenesis, Activity Assay, Incubation

    F. novicida BplA is required for replication in macrophages. (A) Murine bone marrow-derived macrophages were infected with a 20:1 MOI of wild-type F. novicida U112 (WT) and the Δ bplA , Δ birA , bplA complemented (comp), and birA complemented (comp) strains. CFU were quantified at 5.5 h postinfection. (B) Murine BMDM macrophages were infected with wild-type F. novicida U112 (MOI 20:1), and expression of bplA and birA was quantified by qRT-PCR relative to expression of the housekeeping gene uvrD at 30 min, 1 h, and 2 h postinfection (hpi). ***, P
    Figure Legend Snippet: F. novicida BplA is required for replication in macrophages. (A) Murine bone marrow-derived macrophages were infected with a 20:1 MOI of wild-type F. novicida U112 (WT) and the Δ bplA , Δ birA , bplA complemented (comp), and birA complemented (comp) strains. CFU were quantified at 5.5 h postinfection. (B) Murine BMDM macrophages were infected with wild-type F. novicida U112 (MOI 20:1), and expression of bplA and birA was quantified by qRT-PCR relative to expression of the housekeeping gene uvrD at 30 min, 1 h, and 2 h postinfection (hpi). ***, P

    Techniques Used: Derivative Assay, Infection, Expressing, Quantitative RT-PCR

    Enzymatic activities of the F. novicida BplA and BirA proteins. (A) SDS-PAGE analysis of the proteins used in these experiments. M, molecular mass standards. The SDS-PAGE results are consistent with the calculated molecular masses of the hexahistidine-tagged versions of E. coli BirA, F. novicida BirA, and F. novicida BplA, which are 36.1, 38.7, and 32.5 kDa, respectively. (B) TLC assays of biotin protein ligase activities. The synthesis of 32 P-labeled biotinoyl-AMP (biotinoyl-adenylate) from [α- 32 P]ATP was assayed as described in Materials and Methods, analyzed by thin-layer chromatography on glass cellulose plates, and visualized by autoradiography. The reaction products biotinoyl-5′-AMP (bio-5′-AMP), ADP, AMP, and the ATP are indicated in the left margin. The proteins assayed are given at the top of the figure. The abilities of the F. novicida proteins to convert α- 32 P-labeled ATP to biotinoyl-AMP and subsequently to AMP were tested by addition of the AccB-87 acceptor protein. (C to F) Low-resolution matrix-assisted laser desorption/ionization analyses of the overall conversion of apo-AccB-87 to the biotinylated species catalyzed by the various ligases in overnight incubations as shown on the panels. The calculated masses of apo-AccB-87 and biotinylated AccB-87 are 9,333.8 and 9,560.1, respectively.
    Figure Legend Snippet: Enzymatic activities of the F. novicida BplA and BirA proteins. (A) SDS-PAGE analysis of the proteins used in these experiments. M, molecular mass standards. The SDS-PAGE results are consistent with the calculated molecular masses of the hexahistidine-tagged versions of E. coli BirA, F. novicida BirA, and F. novicida BplA, which are 36.1, 38.7, and 32.5 kDa, respectively. (B) TLC assays of biotin protein ligase activities. The synthesis of 32 P-labeled biotinoyl-AMP (biotinoyl-adenylate) from [α- 32 P]ATP was assayed as described in Materials and Methods, analyzed by thin-layer chromatography on glass cellulose plates, and visualized by autoradiography. The reaction products biotinoyl-5′-AMP (bio-5′-AMP), ADP, AMP, and the ATP are indicated in the left margin. The proteins assayed are given at the top of the figure. The abilities of the F. novicida proteins to convert α- 32 P-labeled ATP to biotinoyl-AMP and subsequently to AMP were tested by addition of the AccB-87 acceptor protein. (C to F) Low-resolution matrix-assisted laser desorption/ionization analyses of the overall conversion of apo-AccB-87 to the biotinylated species catalyzed by the various ligases in overnight incubations as shown on the panels. The calculated masses of apo-AccB-87 and biotinylated AccB-87 are 9,333.8 and 9,560.1, respectively.

    Techniques Used: SDS Page, Thin Layer Chromatography, Labeling, Autoradiography

    F. novicida BirA represses bioF expression. Expression of bioF in wild-type F. novicida U112 (WT) and the Δ bplA , Δ birA , bplA , and birA trans complemented (comp) strains was measured by quantitative real-time PCR (qRT-PCR) in relation to the housekeeping gene uvrD . **, P
    Figure Legend Snippet: F. novicida BirA represses bioF expression. Expression of bioF in wild-type F. novicida U112 (WT) and the Δ bplA , Δ birA , bplA , and birA trans complemented (comp) strains was measured by quantitative real-time PCR (qRT-PCR) in relation to the housekeeping gene uvrD . **, P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Biotin protein ligase reaction and regulation of E. coli biotin operon transcription. (A) Biotin protein ligase activity of BirA and BplA. (B to D) General model of bio operon regulation by BirA. Pink ovals, BirA; tailed blue ovals, AccB; green dots, biotin; green dots with red pentagons, bio-5′-AMP (biotinoyl-5′-adenylate). Panel B shows the transcriptionally repressed state, whereas panels C and D separately show the two modes of derepression of bio operon transcription engendered by either biotin limitation or excess unbiotinylated AccB acceptor protein. Both derepression modes act by decreasing the level of biotinoyl-AMP, the ligand required for operator binding by BirA.
    Figure Legend Snippet: Biotin protein ligase reaction and regulation of E. coli biotin operon transcription. (A) Biotin protein ligase activity of BirA and BplA. (B to D) General model of bio operon regulation by BirA. Pink ovals, BirA; tailed blue ovals, AccB; green dots, biotin; green dots with red pentagons, bio-5′-AMP (biotinoyl-5′-adenylate). Panel B shows the transcriptionally repressed state, whereas panels C and D separately show the two modes of derepression of bio operon transcription engendered by either biotin limitation or excess unbiotinylated AccB acceptor protein. Both derepression modes act by decreasing the level of biotinoyl-AMP, the ligand required for operator binding by BirA.

    Techniques Used: Activity Assay, Activated Clotting Time Assay, Binding Assay

    25) Product Images from "Exploration of New Sites in Adenovirus Hexon for Foreign Peptides Insertion"

    Article Title: Exploration of New Sites in Adenovirus Hexon for Foreign Peptides Insertion

    Journal: The Open Virology Journal

    doi: 10.2174/1874357901509010001

    Sites in the HVRs that were modified with a peptide containing the PRRSV major neutralizing epitope B. The arrows mark the position where the peptide was inserted. The numbers show the positions of the amino acid residues in hAd5 hexon. Peptide sequence corresponding to the PRRSV epitope is underlined.
    Figure Legend Snippet: Sites in the HVRs that were modified with a peptide containing the PRRSV major neutralizing epitope B. The arrows mark the position where the peptide was inserted. The numbers show the positions of the amino acid residues in hAd5 hexon. Peptide sequence corresponding to the PRRSV epitope is underlined.

    Techniques Used: Modification, Sequencing

    26) Product Images from "Exploration of New Sites in Adenovirus Hexon for Foreign Peptides Insertion"

    Article Title: Exploration of New Sites in Adenovirus Hexon for Foreign Peptides Insertion

    Journal: The Open Virology Journal

    doi: 10.2174/1874357901509010001

    The PRRSV epitope incorporated in HVR7 of hexon is accessible in the context of an intact virion. In the assay, varying amounts of purified viruses were immobilized in the wells of ELISA plate and incubated with anti-peptide antibody. The binding was detected with an AP-conjugated secondary antibody. Values are expressed as the mean and standard deviation. * indicates a P value of
    Figure Legend Snippet: The PRRSV epitope incorporated in HVR7 of hexon is accessible in the context of an intact virion. In the assay, varying amounts of purified viruses were immobilized in the wells of ELISA plate and incubated with anti-peptide antibody. The binding was detected with an AP-conjugated secondary antibody. Values are expressed as the mean and standard deviation. * indicates a P value of

    Techniques Used: Purification, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Standard Deviation

    27) Product Images from "Enhanced Arabidopsis pattern-triggered immunity by overexpression of cysteine-rich receptor-like kinases"

    Article Title: Enhanced Arabidopsis pattern-triggered immunity by overexpression of cysteine-rich receptor-like kinases

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2015.00322

    CRK4, CRK6, and CRK36 associate with the PRR FLS2 in a flg22-independent manner. (A) Analysis of associations between FLS2 and CRKs by BiFC assay. Arabidopsis protoplasts were co-transfected with FLS2-YFP N + BAK1-YFP C (positive control) or FLS2-YFP N + CRKs-YFP C plasmids and treated with (+) or without (−) 100 nM flg22 for 20 min. The YFP fluorescence (yellow), chlorophyll autofluorescence (red), bright field and the combined images were visualized under a confocal microscope 16 h after transfection. The scale bar represents 10 μm. (B) Association between RCI2B and CRKs by BiFC assay. Arabidopsis protoplasts were co-transfected with RCI2B-YFP N + RCI2B-YFP C (positive control), or RCI2B-YFP N + CRKs-YFP C plasmids and were observed by confocal microscopy 16 h after transfection. The scale bar represents 10 μm. (C) Analysis of association between FLS2 and CRKs by Co-IP assay. Arabidopsis protoplasts co-expressing RCI2B-GFP + FLS2-HA 3 , pG103 empty vector (EV-GFP) + FLS2-HA 3 , or CRKs-GFP + FLS2-HA 3 constructs were treated with (+) or without (−) 100 nM flg22 for 10 min. Total protein extracts (input) and IP-proteins were detected using immunoblotting with an α-GFP or α-HA antibody. Experiments in (A–C) were repeated three times with similar results.
    Figure Legend Snippet: CRK4, CRK6, and CRK36 associate with the PRR FLS2 in a flg22-independent manner. (A) Analysis of associations between FLS2 and CRKs by BiFC assay. Arabidopsis protoplasts were co-transfected with FLS2-YFP N + BAK1-YFP C (positive control) or FLS2-YFP N + CRKs-YFP C plasmids and treated with (+) or without (−) 100 nM flg22 for 20 min. The YFP fluorescence (yellow), chlorophyll autofluorescence (red), bright field and the combined images were visualized under a confocal microscope 16 h after transfection. The scale bar represents 10 μm. (B) Association between RCI2B and CRKs by BiFC assay. Arabidopsis protoplasts were co-transfected with RCI2B-YFP N + RCI2B-YFP C (positive control), or RCI2B-YFP N + CRKs-YFP C plasmids and were observed by confocal microscopy 16 h after transfection. The scale bar represents 10 μm. (C) Analysis of association between FLS2 and CRKs by Co-IP assay. Arabidopsis protoplasts co-expressing RCI2B-GFP + FLS2-HA 3 , pG103 empty vector (EV-GFP) + FLS2-HA 3 , or CRKs-GFP + FLS2-HA 3 constructs were treated with (+) or without (−) 100 nM flg22 for 10 min. Total protein extracts (input) and IP-proteins were detected using immunoblotting with an α-GFP or α-HA antibody. Experiments in (A–C) were repeated three times with similar results.

    Techniques Used: Bimolecular Fluorescence Complementation Assay, Transfection, Positive Control, Fluorescence, Microscopy, Confocal Microscopy, Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation, Construct

    28) Product Images from "Mos1-Mediated Transgenesis to Probe Consequences of Single Gene Mutations in Variation-Rich Isolates of Caenorhabditis elegans"

    Article Title: Mos1-Mediated Transgenesis to Probe Consequences of Single Gene Mutations in Variation-Rich Isolates of Caenorhabditis elegans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0048762

    A single-copy transgene insertion used to investigate consequences of single gene mutations in variation-rich isolates of C. elegans . ( a ) Analysis of tac-1(or402) using Mos1 -mediated transgenesis. tac-1 , including its 5′ and 3′ regulatory sequences, was amplified using high-fidelity DNA polymerase from EU1004 genomic DNA and cloned into a pCFJ178 vector. The red dotted line located in the third exon of tac-1 depicts the or369/402 A to G change that results in an L229F amino acid change. Once cloned into the pCFJ178 vector, the transgene was inserted into the cxTi10882 Mos1 (depicted in orange) integration site on chromosome IV (depicted in purple). The resulting JNC152 strain contains both the endogenous copy of tac-1 located on chromosome II (depicted in blue), and tac-1 isolated from EU1004 inserted on chromosome IV, dotSi121 . To uncover the effect of tac-1(or402) , dotSi121 was examined in the absence of endogenous TAC-1 using ok3305 . Thus, we constructed JNC153. ( b ) Schematic representation of the method used to investigate consequences of tac-1 variations detected in CB4856 tac-1 , including the 5′ and 3′ regulatory sequences, was amplified using high-fidelity DNA polymerase from CB4856 genomic DNA and cloned into a pCFJ178 vector. The red dotted lines represent single nucleotide changes detected in CB4856 tac-1 . Then, the transgene was inserted into the cxTi10882 Mos1 (depicted in orange) integration site on chromosome IV (depicted in purple). JNC150 contains both the endogenous copy of tac-1 located on chromosome II (depicted in blue) and tac-1 isolated from CB4856 dotSi120 inserted on chromosome IV. Then, dotSi120 was analyzed in the absence of endogenous tac-1(ok3305) . ( c ) PCR bands of the expected size (6kb) for stably integrated single copy insertions of tac-1 , dotSi121 and dotSi120 .
    Figure Legend Snippet: A single-copy transgene insertion used to investigate consequences of single gene mutations in variation-rich isolates of C. elegans . ( a ) Analysis of tac-1(or402) using Mos1 -mediated transgenesis. tac-1 , including its 5′ and 3′ regulatory sequences, was amplified using high-fidelity DNA polymerase from EU1004 genomic DNA and cloned into a pCFJ178 vector. The red dotted line located in the third exon of tac-1 depicts the or369/402 A to G change that results in an L229F amino acid change. Once cloned into the pCFJ178 vector, the transgene was inserted into the cxTi10882 Mos1 (depicted in orange) integration site on chromosome IV (depicted in purple). The resulting JNC152 strain contains both the endogenous copy of tac-1 located on chromosome II (depicted in blue), and tac-1 isolated from EU1004 inserted on chromosome IV, dotSi121 . To uncover the effect of tac-1(or402) , dotSi121 was examined in the absence of endogenous TAC-1 using ok3305 . Thus, we constructed JNC153. ( b ) Schematic representation of the method used to investigate consequences of tac-1 variations detected in CB4856 tac-1 , including the 5′ and 3′ regulatory sequences, was amplified using high-fidelity DNA polymerase from CB4856 genomic DNA and cloned into a pCFJ178 vector. The red dotted lines represent single nucleotide changes detected in CB4856 tac-1 . Then, the transgene was inserted into the cxTi10882 Mos1 (depicted in orange) integration site on chromosome IV (depicted in purple). JNC150 contains both the endogenous copy of tac-1 located on chromosome II (depicted in blue) and tac-1 isolated from CB4856 dotSi120 inserted on chromosome IV. Then, dotSi120 was analyzed in the absence of endogenous tac-1(ok3305) . ( c ) PCR bands of the expected size (6kb) for stably integrated single copy insertions of tac-1 , dotSi121 and dotSi120 .

    Techniques Used: Amplification, Clone Assay, Plasmid Preparation, Isolation, Construct, Polymerase Chain Reaction, Stable Transfection

    29) Product Images from "Recombinase-mediated cassette exchange (RMCE) system for functional genomics studies in Mycoplasma mycoides"

    Article Title: Recombinase-mediated cassette exchange (RMCE) system for functional genomics studies in Mycoplasma mycoides

    Journal: Biological Procedures Online

    doi: 10.1186/s12575-015-0016-8

    Design of the Recombinase-Mediated Cassette Exchange. (A) The scheme of RMCE between the recipient plasmid (pRC59) and the donor plasmid (pRC60). pRC59 contains a floxed cassette, consisting of the truncated 3′URA3 gene and the yeast LEU2 marker; and pRC60 contains the 5′URA3 gene, a floxed yeast MET14 ORF, and the Cre recombinase gene under the GAL1 inducible promoter. The gray color indicates the actin intron. The purple bars represent 34 bp hetero-specific loxP mutants where cassette exchange takes place, marked by broken arrows. The cassette exchange was performed by growing the yeast harboring two plasmids in medium containing galactose for 24 hours, followed by the selection of uracil prototrophs on SD-Uracil plates. The cassette exchange would produce two plasmids, pRC59S and pRC60S. The exchange event was evaluated by PCR using primers (swap-F and swap-R) indicated by red arrows. pRC59S allows the amplification of a 1.1 kb product, in contrast to the 3.6 kb product amplified from the parental pRC59. (B) PCR screening for cassette exchange. Cassette exchange was performed in two yeast strains, W303a and VL6-48. Fifteen colonies from each strain were analyzed by PCR. Lanes 1 to 15: W303a strain; and lanes 16 to 30: VL6-48 strain; M: DNA marker.
    Figure Legend Snippet: Design of the Recombinase-Mediated Cassette Exchange. (A) The scheme of RMCE between the recipient plasmid (pRC59) and the donor plasmid (pRC60). pRC59 contains a floxed cassette, consisting of the truncated 3′URA3 gene and the yeast LEU2 marker; and pRC60 contains the 5′URA3 gene, a floxed yeast MET14 ORF, and the Cre recombinase gene under the GAL1 inducible promoter. The gray color indicates the actin intron. The purple bars represent 34 bp hetero-specific loxP mutants where cassette exchange takes place, marked by broken arrows. The cassette exchange was performed by growing the yeast harboring two plasmids in medium containing galactose for 24 hours, followed by the selection of uracil prototrophs on SD-Uracil plates. The cassette exchange would produce two plasmids, pRC59S and pRC60S. The exchange event was evaluated by PCR using primers (swap-F and swap-R) indicated by red arrows. pRC59S allows the amplification of a 1.1 kb product, in contrast to the 3.6 kb product amplified from the parental pRC59. (B) PCR screening for cassette exchange. Cassette exchange was performed in two yeast strains, W303a and VL6-48. Fifteen colonies from each strain were analyzed by PCR. Lanes 1 to 15: W303a strain; and lanes 16 to 30: VL6-48 strain; M: DNA marker.

    Techniques Used: Plasmid Preparation, Marker, Selection, Polymerase Chain Reaction, Amplification

    30) Product Images from "Cloning, characterization, and expression analysis of the pig (Sus scrofa) C1q tumor necrosis factor-related protein-5 gene"

    Article Title: Cloning, characterization, and expression analysis of the pig (Sus scrofa) C1q tumor necrosis factor-related protein-5 gene

    Journal: Molecular Vision

    doi:

    Genomic structure of p CTRP5 and protein homology of pig CTRP5 with the human protein. A : The p CTRP5 was amplified from genomic DNA by PCR, cloned, and sequenced. The p CTRP5 consists of two exons; its coding sequence is highlighted in yellow, the Ser163 codon is highlighted in red, a potential in-frame alternative start codon is highlighted in pink, the transcriptional start site (TSS) is highlighted in blue, and various transcription factor binding sites are highlighted in green. B : The protein homology between the pCTRP5 and hCTRP5 protein is represented. The pCTRP5 protein sequence is shown in black and the human in blue. The underlined residues indicate the five amino acid differences between the pig and human protein. The Ser shown in red indicates residue 163.
    Figure Legend Snippet: Genomic structure of p CTRP5 and protein homology of pig CTRP5 with the human protein. A : The p CTRP5 was amplified from genomic DNA by PCR, cloned, and sequenced. The p CTRP5 consists of two exons; its coding sequence is highlighted in yellow, the Ser163 codon is highlighted in red, a potential in-frame alternative start codon is highlighted in pink, the transcriptional start site (TSS) is highlighted in blue, and various transcription factor binding sites are highlighted in green. B : The protein homology between the pCTRP5 and hCTRP5 protein is represented. The pCTRP5 protein sequence is shown in black and the human in blue. The underlined residues indicate the five amino acid differences between the pig and human protein. The Ser shown in red indicates residue 163.

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

    Expression and localization of CTRP5 in the pig eye. A : Expression of p CTRP5 was studied by qRT–PCR using total mRNA extracted from different tissues of a 222-day-old pig eye. The p CTRP5 expression is presented as bars using an arbitrary scale on the y axis. Values are presented as mean (±SEM) of three independent observations after normalization with the control gene (HGPRT) B : western blot analysis of CTRP5 protein extracted from a 222 days pig. Retina (lane 1), optic nerve (lane 2), lens (lane 3), RPE (lane 4), ciliary body (lane 5), Choroid (lane 6). Detection with an anti-CTRP5 antibody shows significant expression of pCTRP5 protein in the RPE with an expected size molecular weight of approximately 31 kDa. C : Localization of CTRP5 in retinal sections as evaluated by IHC analysis of retinal sections with human monoclonal anti-CTRP5 antibody and Alexa Fluor 488 staining (green, arrows), nuclei (*) stained with DAPI (blue). D : Localization of CTRP5 in the ciliary body as shown by IHC with human monoclonal anti-CTRP5 antibody and Alexa Fluor 555 (red, arrows), nuclei stained with DAPI (blue).
    Figure Legend Snippet: Expression and localization of CTRP5 in the pig eye. A : Expression of p CTRP5 was studied by qRT–PCR using total mRNA extracted from different tissues of a 222-day-old pig eye. The p CTRP5 expression is presented as bars using an arbitrary scale on the y axis. Values are presented as mean (±SEM) of three independent observations after normalization with the control gene (HGPRT) B : western blot analysis of CTRP5 protein extracted from a 222 days pig. Retina (lane 1), optic nerve (lane 2), lens (lane 3), RPE (lane 4), ciliary body (lane 5), Choroid (lane 6). Detection with an anti-CTRP5 antibody shows significant expression of pCTRP5 protein in the RPE with an expected size molecular weight of approximately 31 kDa. C : Localization of CTRP5 in retinal sections as evaluated by IHC analysis of retinal sections with human monoclonal anti-CTRP5 antibody and Alexa Fluor 488 staining (green, arrows), nuclei (*) stained with DAPI (blue). D : Localization of CTRP5 in the ciliary body as shown by IHC with human monoclonal anti-CTRP5 antibody and Alexa Fluor 555 (red, arrows), nuclei stained with DAPI (blue).

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Molecular Weight, Immunohistochemistry, Staining

    31) Product Images from "Functional classification and biochemical characterization of a novel rho class glutathione S-transferase in Synechocystis PCC 6803"

    Article Title: Functional classification and biochemical characterization of a novel rho class glutathione S-transferase in Synechocystis PCC 6803

    Journal: FEBS Open Bio

    doi: 10.1016/j.fob.2014.11.006

    Phylogenetic tree of GST sequences from different species that are similar to GST-sll1545 of Synechocystis PCC 6803.
    Figure Legend Snippet: Phylogenetic tree of GST sequences from different species that are similar to GST-sll1545 of Synechocystis PCC 6803.

    Techniques Used: Periodic Counter-current Chromatography

    Superposition of the molecular model of sll1545 with a zeta class GST. Homology modeling of sll1545 was performed using swissmodel. The model was superimposed on the structure of human zeta class GST (PDB ID: 1FW1 ). Golden and blue color represents 1FW1 and the sll1545 respectively. Green line-represents structurally aligned or superimposed residues in both. The structures were visualized using UCSF Chimera 1.9. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: Superposition of the molecular model of sll1545 with a zeta class GST. Homology modeling of sll1545 was performed using swissmodel. The model was superimposed on the structure of human zeta class GST (PDB ID: 1FW1 ). Golden and blue color represents 1FW1 and the sll1545 respectively. Green line-represents structurally aligned or superimposed residues in both. The structures were visualized using UCSF Chimera 1.9. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used:

    Overexpression of sll1545 in E. coli and purification of the recombinant protein on Ni–NTA agarose. (A) SDS–PAGE analysis of cell lysate showing overexpression of sll1545 and the purified protein. Lanes 1–4 represent molecular weight markers, supernatant of un-induced culture lysate, supernatant of induced culture lysate and purified protein, respectively. (B) Molecular weight and subunit structure of sll1545. SEC profile of sll1545 on Superdex™ 200 10/300 GL column at pH 8.0 and 25 °C.
    Figure Legend Snippet: Overexpression of sll1545 in E. coli and purification of the recombinant protein on Ni–NTA agarose. (A) SDS–PAGE analysis of cell lysate showing overexpression of sll1545 and the purified protein. Lanes 1–4 represent molecular weight markers, supernatant of un-induced culture lysate, supernatant of induced culture lysate and purified protein, respectively. (B) Molecular weight and subunit structure of sll1545. SEC profile of sll1545 on Superdex™ 200 10/300 GL column at pH 8.0 and 25 °C.

    Techniques Used: Over Expression, Purification, Recombinant, SDS Page, Molecular Weight, Size-exclusion Chromatography

    Effect of pH and temperature on the enzymatic activity of sll1545. (A) Effect of pH on the catalytic activity of sll1545. (B) Effect of temperature on the activity of sll1545.
    Figure Legend Snippet: Effect of pH and temperature on the enzymatic activity of sll1545. (A) Effect of pH on the catalytic activity of sll1545. (B) Effect of temperature on the activity of sll1545.

    Techniques Used: Activity Assay

    Multiple amino acid sequence alignment. Synechocystis PCC 6803 sll1545 (P74665) and Arabidiopsis thaliana zeta GST (Q9ZVQ3) were aligned using ESpript 3.0 software utilizing the clustalW algorithm. Similar residues are shown in yellow boxes, red boxes represent identical amino acid residues while residues having different property are without boxes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: Multiple amino acid sequence alignment. Synechocystis PCC 6803 sll1545 (P74665) and Arabidiopsis thaliana zeta GST (Q9ZVQ3) were aligned using ESpript 3.0 software utilizing the clustalW algorithm. Similar residues are shown in yellow boxes, red boxes represent identical amino acid residues while residues having different property are without boxes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: Sequencing, Periodic Counter-current Chromatography, Software

    32) Product Images from "Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells"

    Article Title: Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003666

    rEtpE-C-coated beads enter macrophages by a pathway similar to one that mediates E. chaffeensis entry. (A) Latex beads (red) coated with rEtpE-C by anti-EtpE-C labeling (green) under fluorescence microscopy. Scale bar, 1 µm. (B) Fluorescence and phase contrast merged images of rEtpE-C-coated beads incubated with mouse BMDMs. Cells were pretreated with DMSO (solvent control), MDC, genistein, or PI-PLC for 45 min followed by trypsin treatment to remove beads that were not internalized. Scale bar, 10 µm. (C and D) Numbers of internalized rEtpE-C-coated (C) and non-coated (D) beads/cell incubated with mouse BMDMs pretreated with MDC, genistein, or PI-PLC, relative to DMSO treatment (solvent control) set as 100. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P
    Figure Legend Snippet: rEtpE-C-coated beads enter macrophages by a pathway similar to one that mediates E. chaffeensis entry. (A) Latex beads (red) coated with rEtpE-C by anti-EtpE-C labeling (green) under fluorescence microscopy. Scale bar, 1 µm. (B) Fluorescence and phase contrast merged images of rEtpE-C-coated beads incubated with mouse BMDMs. Cells were pretreated with DMSO (solvent control), MDC, genistein, or PI-PLC for 45 min followed by trypsin treatment to remove beads that were not internalized. Scale bar, 10 µm. (C and D) Numbers of internalized rEtpE-C-coated (C) and non-coated (D) beads/cell incubated with mouse BMDMs pretreated with MDC, genistein, or PI-PLC, relative to DMSO treatment (solvent control) set as 100. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P

    Techniques Used: Labeling, Fluorescence, Microscopy, Incubation, Planar Chromatography, Standard Deviation

    EtpE-C binds DNase X. (A) Far-Western blotting of renatured rEtpE-C and rECH0825 on a nitrocellulose membrane incubated with THP-1 cell lysate. Native DNase X was detected with anti-DNase X (α-DNase X), and recombinant proteins were detected with anti-histidine-tag (α-His tag). (B) Western blotting of THP-1 cell lysate following affinity pull-down with rEtpE-C bound to Ni-silica matrix. Bound proteins were eluted with imidazole and labeled with α-DNase X or α-His tag. (C) Western blot analysis of E. chaffeensis- infected THP-1 cell lysate immunoprecipitated with anti-EtpE-C (α-EtpE-C) or control IgG. THP-1 cells were incubated with E. chaffeensis for 30 min, followed by lysis, and immunoprecipitated with α-EtpE-C- or control mouse IgG-bound protein A agarose. The precipitates were subjected to Western blotting with α-DNase X. ** DNase X, * mouse IgG heavy chain. (D) Immunofluorescence labeling of rEtpE-C-coated latex beads (red) incubated with RF/6A cells for 1 h with α-DNase X without permeabilization. Note a cluster of beads colocalizes with host cell-surface DNase X. Scale bar, 5 µm. (E) Selected time-lapse images (0 to 6:38 min) of rEtpE-C-coated beads attached to RF/6A cells expressing DNase X-GFP at 4°C, and time 0 min was set upon raising the temperature to 37°C. The white dashed line denotes the RF/6A cell contour. A single z- plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 2 µm (see also Movie S1 ). (F) Line intensity profile analysis of red (rEtpE-C-beads) and green (DNase X-GFP) signal along the length of the line (slanted white line in the image 5E).
    Figure Legend Snippet: EtpE-C binds DNase X. (A) Far-Western blotting of renatured rEtpE-C and rECH0825 on a nitrocellulose membrane incubated with THP-1 cell lysate. Native DNase X was detected with anti-DNase X (α-DNase X), and recombinant proteins were detected with anti-histidine-tag (α-His tag). (B) Western blotting of THP-1 cell lysate following affinity pull-down with rEtpE-C bound to Ni-silica matrix. Bound proteins were eluted with imidazole and labeled with α-DNase X or α-His tag. (C) Western blot analysis of E. chaffeensis- infected THP-1 cell lysate immunoprecipitated with anti-EtpE-C (α-EtpE-C) or control IgG. THP-1 cells were incubated with E. chaffeensis for 30 min, followed by lysis, and immunoprecipitated with α-EtpE-C- or control mouse IgG-bound protein A agarose. The precipitates were subjected to Western blotting with α-DNase X. ** DNase X, * mouse IgG heavy chain. (D) Immunofluorescence labeling of rEtpE-C-coated latex beads (red) incubated with RF/6A cells for 1 h with α-DNase X without permeabilization. Note a cluster of beads colocalizes with host cell-surface DNase X. Scale bar, 5 µm. (E) Selected time-lapse images (0 to 6:38 min) of rEtpE-C-coated beads attached to RF/6A cells expressing DNase X-GFP at 4°C, and time 0 min was set upon raising the temperature to 37°C. The white dashed line denotes the RF/6A cell contour. A single z- plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 2 µm (see also Movie S1 ). (F) Line intensity profile analysis of red (rEtpE-C-beads) and green (DNase X-GFP) signal along the length of the line (slanted white line in the image 5E).

    Techniques Used: Far Western Blot, Incubation, Recombinant, Western Blot, Labeling, Infection, Immunoprecipitation, Lysis, Immunofluorescence, Expressing, Microscopy

    Schematic representation of E. chaffeensis binding and entry into mammalian cells. DNase X is enriched in the lipid raft domains of the cell membrane. Extracellular E. chaffeensis uses its surface protein EtpE C-terminal region to make initial contacts with cell surface DNase X that results in further lateral redistribution and local clustering of DNaseX at the sites of bacterial binding. This binding elicits signals that are relayed down-stream and culminated in host cytoskeletal remodeling, filopodial induction and engulfment of the bound bacteria into an early endosome into the host cell. This receptor-mediated endocytosis can be specifically disrupted by genistein, verapamil or MDC. Latex beads coated with rEtpE-C also bind to cell surface DNase X and follows a similar pattern of entry like that of E. chaffeensis .
    Figure Legend Snippet: Schematic representation of E. chaffeensis binding and entry into mammalian cells. DNase X is enriched in the lipid raft domains of the cell membrane. Extracellular E. chaffeensis uses its surface protein EtpE C-terminal region to make initial contacts with cell surface DNase X that results in further lateral redistribution and local clustering of DNaseX at the sites of bacterial binding. This binding elicits signals that are relayed down-stream and culminated in host cytoskeletal remodeling, filopodial induction and engulfment of the bound bacteria into an early endosome into the host cell. This receptor-mediated endocytosis can be specifically disrupted by genistein, verapamil or MDC. Latex beads coated with rEtpE-C also bind to cell surface DNase X and follows a similar pattern of entry like that of E. chaffeensis .

    Techniques Used: Binding Assay

    EtpE is expressed by E. chaffeensis in HME patients and infected dogs, and immunization with rEtpE-C protects mice against E. chaffeensis challenge. (A) SDS-PAGE analysis and GelCode Blue staining of rEtpE-N (lane 1) and rEtpE-C (lane 2) (5 µg/lane). rEtpE-N was partially cleaved after its expression in E. coli and thus is visualized as multiple bands. (B) Western blot analysis of rEtpE-N (lane 1) and rEtpE-C (lane 2) (5 µg/lane) with HME patient sera (ID: 72088, MRL1-22, MRL1-40) or control human serum (Control), or sera from dogs experimentally infected with E. chaffeensis (ID: CTUALJ, 3918815, 1425) or control dog serum. The relative band intensity for rEtpE-N/rEtpE-C (75 kDa and 34 kDa bands) assessed by densitometry was shown beneath the panels. (C) Dot-plot analysis of E. chaffeensis load of the blood samples from rEtpE-C-immunized and placebo-immunized mice at 5 days after E. chaffeensis challenge. qPCR of E. chaffeensis 16S rDNA normalized to mouse G3PDH DNA. *Significantly different ( P
    Figure Legend Snippet: EtpE is expressed by E. chaffeensis in HME patients and infected dogs, and immunization with rEtpE-C protects mice against E. chaffeensis challenge. (A) SDS-PAGE analysis and GelCode Blue staining of rEtpE-N (lane 1) and rEtpE-C (lane 2) (5 µg/lane). rEtpE-N was partially cleaved after its expression in E. coli and thus is visualized as multiple bands. (B) Western blot analysis of rEtpE-N (lane 1) and rEtpE-C (lane 2) (5 µg/lane) with HME patient sera (ID: 72088, MRL1-22, MRL1-40) or control human serum (Control), or sera from dogs experimentally infected with E. chaffeensis (ID: CTUALJ, 3918815, 1425) or control dog serum. The relative band intensity for rEtpE-N/rEtpE-C (75 kDa and 34 kDa bands) assessed by densitometry was shown beneath the panels. (C) Dot-plot analysis of E. chaffeensis load of the blood samples from rEtpE-C-immunized and placebo-immunized mice at 5 days after E. chaffeensis challenge. qPCR of E. chaffeensis 16S rDNA normalized to mouse G3PDH DNA. *Significantly different ( P

    Techniques Used: Infection, Mouse Assay, SDS Page, Staining, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    rEtpE-C-coated latex beads bind and enter non-phagocytic host cells. (A) rEtpE-C-coated beads (arrows), but not rEtpE-N, rECH0825, or rGroEL-coated beads, bind and enter HEK293 cells at 1 h pi. Scale bar, 10 µm. (B) Bar graph showing quantitation of similar experiment as (A) by scoring beads in 100 cells. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P
    Figure Legend Snippet: rEtpE-C-coated latex beads bind and enter non-phagocytic host cells. (A) rEtpE-C-coated beads (arrows), but not rEtpE-N, rECH0825, or rGroEL-coated beads, bind and enter HEK293 cells at 1 h pi. Scale bar, 10 µm. (B) Bar graph showing quantitation of similar experiment as (A) by scoring beads in 100 cells. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P

    Techniques Used: Quantitation Assay, Standard Deviation

    EtpE-C is exposed at the bacterial surface, and anti-EtpE-C neutralizes E. chaffeensis infection in vitro . (A) Western blot analysis of E. chaffeensis- infected ( Ech ) and uninfected DH82 cells at 60 h pi using anti-EtpE-N (α-EtpE-N) and anti-EtpE-C (α-EtpE-C). (B) Double immunofluorescence labeling of E. chaffeensis- infected human primary macrophages derived from peripheral blood monocytes at 56 h pi. Cells were fixed with PFA, permeabilized with saponin, and labeled with anti-EtpE-C and anti- E. chaffeensis major outer membrane protein P28. The white dashed line denotes the macrophage contour. The boxed region indicates the area enlarged in the smaller panels to the right. Merge/DIC: Fluorescence images merged with Differential interference contrast image (DIC). A single z -plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 2 µm. (C) E. chaffeensis was incubated with DH82 cells for 30 min and double immunofluorescence labeling was performed using anti-EtpE-C and anti- E. chaffeensis P28 without permeabilization. DAPI was used to label DNA. Scale bar, 1 µm (see also suppl. Fig. S2 ). (D) Numbers of E. chaffeensis bound to RF/6A cells at 30 min pi. Host cell-free E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and incubated with RF/6A cells for 30 min. Unbound E. chaffeensis was washed away, cells were fixed with PFA, and E. chaffeensis labeled with anti-P28 without permeabilization. E. chaffeensis in 100 cells were scored. (E) Numbers of E. chaffeensis internalized into RF/6A cells at 2 h pi. E. chaffeensis was pretreated with anti-rEtpE-C or preimmune mouse serum and incubated with RF/6A cells for 2 h. To distinguish intracellular from bound E. chaffeensis , unbound E. chaffeensis was washed away and cells were processed for two rounds of immunostaining with anti-P28; first without permeabilization to detect bound but not internalized E. chaffeensis (AF555–conjugated secondary antibody) and second round with saponin permeabilization to detect total E. chaffeensis , i.e., bound plus internalized (AF488–conjugated secondary antibody). E. chaffeensis in 100 cells was scored. The black bar represents total E. chaffeensis and the white bar represents internalized E. chaffeensis (total minus bound) (see also suppl. Fig. S3 ). (F) Infection of RF/6A cells with E. chaffeensis at 48 h pi. E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and used to infect RF/6A cells; cells were harvested at 48 h pi. qPCR for E. chaffeensis 16S rDNA was normalized with G3PDH DNA. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P
    Figure Legend Snippet: EtpE-C is exposed at the bacterial surface, and anti-EtpE-C neutralizes E. chaffeensis infection in vitro . (A) Western blot analysis of E. chaffeensis- infected ( Ech ) and uninfected DH82 cells at 60 h pi using anti-EtpE-N (α-EtpE-N) and anti-EtpE-C (α-EtpE-C). (B) Double immunofluorescence labeling of E. chaffeensis- infected human primary macrophages derived from peripheral blood monocytes at 56 h pi. Cells were fixed with PFA, permeabilized with saponin, and labeled with anti-EtpE-C and anti- E. chaffeensis major outer membrane protein P28. The white dashed line denotes the macrophage contour. The boxed region indicates the area enlarged in the smaller panels to the right. Merge/DIC: Fluorescence images merged with Differential interference contrast image (DIC). A single z -plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 2 µm. (C) E. chaffeensis was incubated with DH82 cells for 30 min and double immunofluorescence labeling was performed using anti-EtpE-C and anti- E. chaffeensis P28 without permeabilization. DAPI was used to label DNA. Scale bar, 1 µm (see also suppl. Fig. S2 ). (D) Numbers of E. chaffeensis bound to RF/6A cells at 30 min pi. Host cell-free E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and incubated with RF/6A cells for 30 min. Unbound E. chaffeensis was washed away, cells were fixed with PFA, and E. chaffeensis labeled with anti-P28 without permeabilization. E. chaffeensis in 100 cells were scored. (E) Numbers of E. chaffeensis internalized into RF/6A cells at 2 h pi. E. chaffeensis was pretreated with anti-rEtpE-C or preimmune mouse serum and incubated with RF/6A cells for 2 h. To distinguish intracellular from bound E. chaffeensis , unbound E. chaffeensis was washed away and cells were processed for two rounds of immunostaining with anti-P28; first without permeabilization to detect bound but not internalized E. chaffeensis (AF555–conjugated secondary antibody) and second round with saponin permeabilization to detect total E. chaffeensis , i.e., bound plus internalized (AF488–conjugated secondary antibody). E. chaffeensis in 100 cells was scored. The black bar represents total E. chaffeensis and the white bar represents internalized E. chaffeensis (total minus bound) (see also suppl. Fig. S3 ). (F) Infection of RF/6A cells with E. chaffeensis at 48 h pi. E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and used to infect RF/6A cells; cells were harvested at 48 h pi. qPCR for E. chaffeensis 16S rDNA was normalized with G3PDH DNA. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P

    Techniques Used: Infection, In Vitro, Western Blot, Immunofluorescence, Labeling, Derivative Assay, Fluorescence, Microscopy, Incubation, Immunostaining, Real-time Polymerase Chain Reaction, Standard Deviation

    Internalization of rEtpE-C-coated beads is dependent on DNase X. (A) Immunofluorescence labeling of rEtpE-C-coated or non-coated beads incubated with human macrophages derived from peripheral blood monocytes. At 30 min pi, cells were labeled with α-DNase X without permeabilization. rEtpE-C-coated beads cluster and colocalize with DNase X on the cell surface, but non-coated beads do not. A single z- plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 5 µm (see also suppl. Fig. S7 and suppl. Movie S2 and S3 ). (B) A selected image showing the orthogonal view of macrophage incubated with rEtpE-C-coated (left panel) or non-coated (right panel) beads in (A). The orthogonal view was obtained from the reconstituted 3-D view of serial z -stack images (combined z-section width of 7.2 µm). Scale bar, 5 µm. The fluorescence intensity profiles of green (DNase X) and red (beads) signals were shown. (C) Fluorescence and phase contrast merged images of rEtpE-C-coated and non-coated beads incubated with BMDMs from DNase X −/− and wild-type mice. Cells and beads were incubated for 45 min followed by trypsin treatment to remove non-internalized beads. Scale bar, 10 µm. (D) Numbers of internalized rEtpE-C-coated beads/cell of similar experiment as (C), relative to the number of non-coated beads set as 100. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P
    Figure Legend Snippet: Internalization of rEtpE-C-coated beads is dependent on DNase X. (A) Immunofluorescence labeling of rEtpE-C-coated or non-coated beads incubated with human macrophages derived from peripheral blood monocytes. At 30 min pi, cells were labeled with α-DNase X without permeabilization. rEtpE-C-coated beads cluster and colocalize with DNase X on the cell surface, but non-coated beads do not. A single z- plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 5 µm (see also suppl. Fig. S7 and suppl. Movie S2 and S3 ). (B) A selected image showing the orthogonal view of macrophage incubated with rEtpE-C-coated (left panel) or non-coated (right panel) beads in (A). The orthogonal view was obtained from the reconstituted 3-D view of serial z -stack images (combined z-section width of 7.2 µm). Scale bar, 5 µm. The fluorescence intensity profiles of green (DNase X) and red (beads) signals were shown. (C) Fluorescence and phase contrast merged images of rEtpE-C-coated and non-coated beads incubated with BMDMs from DNase X −/− and wild-type mice. Cells and beads were incubated for 45 min followed by trypsin treatment to remove non-internalized beads. Scale bar, 10 µm. (D) Numbers of internalized rEtpE-C-coated beads/cell of similar experiment as (C), relative to the number of non-coated beads set as 100. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P

    Techniques Used: Immunofluorescence, Labeling, Incubation, Derivative Assay, Microscopy, Fluorescence, Mouse Assay, Standard Deviation

    33) Product Images from "Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells"

    Article Title: Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003666

    EtpE-C is exposed at the bacterial surface, and anti-EtpE-C neutralizes E. chaffeensis infection in vitro . (A) Western blot analysis of E. chaffeensis- infected ( Ech ) and uninfected DH82 cells at 60 h pi using anti-EtpE-N (α-EtpE-N) and anti-EtpE-C (α-EtpE-C). (B) Double immunofluorescence labeling of E. chaffeensis- infected human primary macrophages derived from peripheral blood monocytes at 56 h pi. Cells were fixed with PFA, permeabilized with saponin, and labeled with anti-EtpE-C and anti- E. chaffeensis major outer membrane protein P28. The white dashed line denotes the macrophage contour. The boxed region indicates the area enlarged in the smaller panels to the right. Merge/DIC: Fluorescence images merged with Differential interference contrast image (DIC). A single z -plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 2 µm. (C) E. chaffeensis was incubated with DH82 cells for 30 min and double immunofluorescence labeling was performed using anti-EtpE-C and anti- E. chaffeensis P28 without permeabilization. DAPI was used to label DNA. Scale bar, 1 µm (see also suppl. Fig. S2 ). (D) Numbers of E. chaffeensis bound to RF/6A cells at 30 min pi. Host cell-free E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and incubated with RF/6A cells for 30 min. Unbound E. chaffeensis was washed away, cells were fixed with PFA, and E. chaffeensis labeled with anti-P28 without permeabilization. E. chaffeensis in 100 cells were scored. (E) Numbers of E. chaffeensis internalized into RF/6A cells at 2 h pi. E. chaffeensis was pretreated with anti-rEtpE-C or preimmune mouse serum and incubated with RF/6A cells for 2 h. To distinguish intracellular from bound E. chaffeensis , unbound E. chaffeensis was washed away and cells were processed for two rounds of immunostaining with anti-P28; first without permeabilization to detect bound but not internalized E. chaffeensis (AF555–conjugated secondary antibody) and second round with saponin permeabilization to detect total E. chaffeensis , i.e., bound plus internalized (AF488–conjugated secondary antibody). E. chaffeensis in 100 cells was scored. The black bar represents total E. chaffeensis and the white bar represents internalized E. chaffeensis (total minus bound) (see also suppl. Fig. S3 ). (F) Infection of RF/6A cells with E. chaffeensis at 48 h pi. E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and used to infect RF/6A cells; cells were harvested at 48 h pi. qPCR for E. chaffeensis 16S rDNA was normalized with G3PDH DNA. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P
    Figure Legend Snippet: EtpE-C is exposed at the bacterial surface, and anti-EtpE-C neutralizes E. chaffeensis infection in vitro . (A) Western blot analysis of E. chaffeensis- infected ( Ech ) and uninfected DH82 cells at 60 h pi using anti-EtpE-N (α-EtpE-N) and anti-EtpE-C (α-EtpE-C). (B) Double immunofluorescence labeling of E. chaffeensis- infected human primary macrophages derived from peripheral blood monocytes at 56 h pi. Cells were fixed with PFA, permeabilized with saponin, and labeled with anti-EtpE-C and anti- E. chaffeensis major outer membrane protein P28. The white dashed line denotes the macrophage contour. The boxed region indicates the area enlarged in the smaller panels to the right. Merge/DIC: Fluorescence images merged with Differential interference contrast image (DIC). A single z -plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 2 µm. (C) E. chaffeensis was incubated with DH82 cells for 30 min and double immunofluorescence labeling was performed using anti-EtpE-C and anti- E. chaffeensis P28 without permeabilization. DAPI was used to label DNA. Scale bar, 1 µm (see also suppl. Fig. S2 ). (D) Numbers of E. chaffeensis bound to RF/6A cells at 30 min pi. Host cell-free E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and incubated with RF/6A cells for 30 min. Unbound E. chaffeensis was washed away, cells were fixed with PFA, and E. chaffeensis labeled with anti-P28 without permeabilization. E. chaffeensis in 100 cells were scored. (E) Numbers of E. chaffeensis internalized into RF/6A cells at 2 h pi. E. chaffeensis was pretreated with anti-rEtpE-C or preimmune mouse serum and incubated with RF/6A cells for 2 h. To distinguish intracellular from bound E. chaffeensis , unbound E. chaffeensis was washed away and cells were processed for two rounds of immunostaining with anti-P28; first without permeabilization to detect bound but not internalized E. chaffeensis (AF555–conjugated secondary antibody) and second round with saponin permeabilization to detect total E. chaffeensis , i.e., bound plus internalized (AF488–conjugated secondary antibody). E. chaffeensis in 100 cells was scored. The black bar represents total E. chaffeensis and the white bar represents internalized E. chaffeensis (total minus bound) (see also suppl. Fig. S3 ). (F) Infection of RF/6A cells with E. chaffeensis at 48 h pi. E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and used to infect RF/6A cells; cells were harvested at 48 h pi. qPCR for E. chaffeensis 16S rDNA was normalized with G3PDH DNA. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P

    Techniques Used: Infection, In Vitro, Western Blot, Immunofluorescence, Labeling, Derivative Assay, Fluorescence, Microscopy, Incubation, Immunostaining, Real-time Polymerase Chain Reaction, Standard Deviation

    34) Product Images from "LESR2 is a lymphatic endothelial-specific lncRNA that governs cell proliferation and migration through KLF4 and SEMA3C"

    Article Title: LESR2 is a lymphatic endothelial-specific lncRNA that governs cell proliferation and migration through KLF4 and SEMA3C

    Journal: bioRxiv

    doi: 10.1101/2020.05.25.114546

    LESR2 is a nuclear lncRNA interacting in trans with DNA regions near a subset of differentially expressed genes. (a) Representative images of negative control dapB (bacterial gene), MALAT-1 (nuclear lncRNA), and LESR2 expression using smRNA-FISH. Immunostaining of endothelial cell marker CD31 was used to outline cell shape. Scale bars represent 20 μ m. (b) Quantification of the nuclear (green) and cytoplasmic (black) smRNA-FISH signal of LESR2 in neonatal LECs derived from 2 donors quantified with ImageJ 97 . Bars represent nuclear and cytoplasmic percentages displayed as mean + SD. (c) Pie chart showing the genomic localization of 2,258 LESR2 peaks in protein-coding, overlap between protein-coding and noncoding, noncoding, and intergenic regions according to FANTOM CAT annotations using bedtools 103 . Magnification shows the distribution of LESR2 binding sites within promoter, exon, or intron of protein-coding genes (1,607 genes). LESR2 peaks are listed in Supplementary Table 9. (d) Venn diagrams showing the overlap between total genes expressed in LECs (TPM and CPM > 0.5) and identified LESR2-ChIRP genes, and the significant overlap between LESR2-ChIRP genes and differentially expressed genes after LESR2-ASOKD. (e) Circular plot showing genome-wide interactions of LESR2 near the 44 targets generated by Circos 104 . Scaled chromosomes with their respective cytobands are placed in circle. Major and minor ticks represent 50Mb and 10Mb, respectively. Orange and purple lines show interactions between LESR2 locus and its up- and downregulated targets, respectively. Green line highlights the genomic locus of LESR2. (f) Heat maps based on expression levels (CAGE-Seq, CPM) in control ASO and LESR2-ASOKD samples (up, 2 replicates), as well as in LECs and BECs (down, 2 replicates) of the 44 LESR2 targets (orange: upregulated, purple: downregulated). Color code for row Z-Score values on a scale from −1 to +1. Genes were ordered by log2FC values of ASOKD data and according to their differential expression between LECs and BECs.
    Figure Legend Snippet: LESR2 is a nuclear lncRNA interacting in trans with DNA regions near a subset of differentially expressed genes. (a) Representative images of negative control dapB (bacterial gene), MALAT-1 (nuclear lncRNA), and LESR2 expression using smRNA-FISH. Immunostaining of endothelial cell marker CD31 was used to outline cell shape. Scale bars represent 20 μ m. (b) Quantification of the nuclear (green) and cytoplasmic (black) smRNA-FISH signal of LESR2 in neonatal LECs derived from 2 donors quantified with ImageJ 97 . Bars represent nuclear and cytoplasmic percentages displayed as mean + SD. (c) Pie chart showing the genomic localization of 2,258 LESR2 peaks in protein-coding, overlap between protein-coding and noncoding, noncoding, and intergenic regions according to FANTOM CAT annotations using bedtools 103 . Magnification shows the distribution of LESR2 binding sites within promoter, exon, or intron of protein-coding genes (1,607 genes). LESR2 peaks are listed in Supplementary Table 9. (d) Venn diagrams showing the overlap between total genes expressed in LECs (TPM and CPM > 0.5) and identified LESR2-ChIRP genes, and the significant overlap between LESR2-ChIRP genes and differentially expressed genes after LESR2-ASOKD. (e) Circular plot showing genome-wide interactions of LESR2 near the 44 targets generated by Circos 104 . Scaled chromosomes with their respective cytobands are placed in circle. Major and minor ticks represent 50Mb and 10Mb, respectively. Orange and purple lines show interactions between LESR2 locus and its up- and downregulated targets, respectively. Green line highlights the genomic locus of LESR2. (f) Heat maps based on expression levels (CAGE-Seq, CPM) in control ASO and LESR2-ASOKD samples (up, 2 replicates), as well as in LECs and BECs (down, 2 replicates) of the 44 LESR2 targets (orange: upregulated, purple: downregulated). Color code for row Z-Score values on a scale from −1 to +1. Genes were ordered by log2FC values of ASOKD data and according to their differential expression between LECs and BECs.

    Techniques Used: Negative Control, Expressing, Fluorescence In Situ Hybridization, Immunostaining, Marker, Derivative Assay, Binding Assay, Genome Wide, Generated, Allele-specific Oligonucleotide

    LESR2 interacts with several protein complexes to exert its regulatory function on gene expression. (a) Protein-protein interaction network using STRING 58 of the 59 identified protein targets after in vitro biotin-RNA pull-down followed by mass spectrometry of LESR2 transcript and its antisense control in neonatal LECs derived from the same donor (n = 2). Proteins were clustered by Markov clustering (MCL) algorithm 107 with an inflation parameter of 1.5. Circles highlight the most relevant clusters. Disconnected nodes were hidden to improve visualization. Lines indicate interactions within each complex. Thickness of the lines indicates the confidence of interaction from text mining, databases, experiments, and co-expression. Interacting proteins are listed in Supplementary Table 10. (b) Graph showing logFC LECs versus BECs against average unique peptide after LESR2 RNA pull-down. Blue lines show the logFC > 0.5 and unique peptide > 5 thresholds. (c) Representative western blot after RNA immunoprecipitation for RBBP7 followed by qPCR for LESR2 in neonatal LECs. To prevent masking from IgG heavy chain, a conformation-specific IgG secondary antibody was used. Uncropped western blot image is shown in Supplementary Figure 8. (d) LESR2 enrichment displayed as FC against IgG Control after RNA immunoprecipitation for RBBP7 in neonatal LECs derived from the same donor. LESR2 qPCR levels were normalized to the housekeeping gene GAPDH. Bars represent mean ± SD (n = 3). *P
    Figure Legend Snippet: LESR2 interacts with several protein complexes to exert its regulatory function on gene expression. (a) Protein-protein interaction network using STRING 58 of the 59 identified protein targets after in vitro biotin-RNA pull-down followed by mass spectrometry of LESR2 transcript and its antisense control in neonatal LECs derived from the same donor (n = 2). Proteins were clustered by Markov clustering (MCL) algorithm 107 with an inflation parameter of 1.5. Circles highlight the most relevant clusters. Disconnected nodes were hidden to improve visualization. Lines indicate interactions within each complex. Thickness of the lines indicates the confidence of interaction from text mining, databases, experiments, and co-expression. Interacting proteins are listed in Supplementary Table 10. (b) Graph showing logFC LECs versus BECs against average unique peptide after LESR2 RNA pull-down. Blue lines show the logFC > 0.5 and unique peptide > 5 thresholds. (c) Representative western blot after RNA immunoprecipitation for RBBP7 followed by qPCR for LESR2 in neonatal LECs. To prevent masking from IgG heavy chain, a conformation-specific IgG secondary antibody was used. Uncropped western blot image is shown in Supplementary Figure 8. (d) LESR2 enrichment displayed as FC against IgG Control after RNA immunoprecipitation for RBBP7 in neonatal LECs derived from the same donor. LESR2 qPCR levels were normalized to the housekeeping gene GAPDH. Bars represent mean ± SD (n = 3). *P

    Techniques Used: Expressing, In Vitro, Mass Spectrometry, Derivative Assay, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction

    Knockdown of LESR2 reduces cell growth, cell cycle progression, and migration of LECs in vitro . (a, b) Cell growth profiles and cell growth rates of neonatal LECs over 48h after ASOKD (a) or CRISPRi-KD (b) of LESR2 using IncuCyte. Sample’s confluences were normalized to T 0 . Growth rates were calculated as the slope of linear regression and normalized to control ASO/sgRNA. (c) Representative flow cytometry plots of neonatal LECs after 24h LESR2-ASOKD. Cells were firstly gated with live/dead Zombie staining (upper plots). Resulting living cells were further gated for non-proliferating stages subG0 and G0, and proliferating stages G1, S, G2, and M, using propidium iodide (IP) and Ki-67 (lower plots). (d) Quantification of the cell cycle progression analysis of neonatal LECs after 24h LESR2-ASOKD. Bars represent percentages of gated living cells in subG0, G0, G1, S, G2, and M. Statistical analysis was performed on G0 populations. (e) Representative images of the wound closure assay (9h) in neonatal LECs after LESR2-ASOKD. Confluence mask is shown for all time points. Before scratch, cells were incubated for 2h with 2 μ g/mL Mitomycin C (proliferation inhibitor) at 37°C. Scale bar represents 200 μ m. (f) Quantification of the wound closure assay (up to 9h) of neonatal LECs after LESR2-ASOKD. Percentages were determined for each time point using TScratch 98 . (g) Schematic representation of 3’ RACE results depicting the three LESR2 transcripts expressed in LECs: LESR2-1 (approx. 1,100bp), LESR2-2 (approx. 1,200bp), LESR2-3 (approx. 600bp). RNA-Seq signal was visualized through the Zenbu genome browser 106 . LESR2 transcript sequences are listed in Supplementary Table 7. (h) Comparison of qPCR levels of GAPDH (polyA+), H2BK (polyA-), LESR2-1, LESR2-2, LESR2-3 after cDNA synthesis with either oligodT or random hexamers primers in neonatal LECs derived from 3 donors. (i) Expression of LESR2-1, LESR2-2, and LESR2-3 relative to housekeeping gene GAPDH in neonatal LECs derived from 3 donors. (j) Quantification of the cell cycle progression analysis of pCDH-empty vector (pCDH-EV) and pCDH-LESR2 infected neonatal LECs after 24h LESR2-ASOKD. Statistical analysis was performed on G0 populations. (k) Quantification of the wound closure assay (up to 9h) of pCDH-EV and pCDH-LESR2 infected neonatal LECs after LESR2-ASOKD. Data are displayed as mean + SD (n = 10 in a, f, and k; n = 5 in b; n = 3 in h, i, and j; n = 2 in d). Percentages represent LESR2 knockdown efficiencies after the experiments. **P
    Figure Legend Snippet: Knockdown of LESR2 reduces cell growth, cell cycle progression, and migration of LECs in vitro . (a, b) Cell growth profiles and cell growth rates of neonatal LECs over 48h after ASOKD (a) or CRISPRi-KD (b) of LESR2 using IncuCyte. Sample’s confluences were normalized to T 0 . Growth rates were calculated as the slope of linear regression and normalized to control ASO/sgRNA. (c) Representative flow cytometry plots of neonatal LECs after 24h LESR2-ASOKD. Cells were firstly gated with live/dead Zombie staining (upper plots). Resulting living cells were further gated for non-proliferating stages subG0 and G0, and proliferating stages G1, S, G2, and M, using propidium iodide (IP) and Ki-67 (lower plots). (d) Quantification of the cell cycle progression analysis of neonatal LECs after 24h LESR2-ASOKD. Bars represent percentages of gated living cells in subG0, G0, G1, S, G2, and M. Statistical analysis was performed on G0 populations. (e) Representative images of the wound closure assay (9h) in neonatal LECs after LESR2-ASOKD. Confluence mask is shown for all time points. Before scratch, cells were incubated for 2h with 2 μ g/mL Mitomycin C (proliferation inhibitor) at 37°C. Scale bar represents 200 μ m. (f) Quantification of the wound closure assay (up to 9h) of neonatal LECs after LESR2-ASOKD. Percentages were determined for each time point using TScratch 98 . (g) Schematic representation of 3’ RACE results depicting the three LESR2 transcripts expressed in LECs: LESR2-1 (approx. 1,100bp), LESR2-2 (approx. 1,200bp), LESR2-3 (approx. 600bp). RNA-Seq signal was visualized through the Zenbu genome browser 106 . LESR2 transcript sequences are listed in Supplementary Table 7. (h) Comparison of qPCR levels of GAPDH (polyA+), H2BK (polyA-), LESR2-1, LESR2-2, LESR2-3 after cDNA synthesis with either oligodT or random hexamers primers in neonatal LECs derived from 3 donors. (i) Expression of LESR2-1, LESR2-2, and LESR2-3 relative to housekeeping gene GAPDH in neonatal LECs derived from 3 donors. (j) Quantification of the cell cycle progression analysis of pCDH-empty vector (pCDH-EV) and pCDH-LESR2 infected neonatal LECs after 24h LESR2-ASOKD. Statistical analysis was performed on G0 populations. (k) Quantification of the wound closure assay (up to 9h) of pCDH-EV and pCDH-LESR2 infected neonatal LECs after LESR2-ASOKD. Data are displayed as mean + SD (n = 10 in a, f, and k; n = 5 in b; n = 3 in h, i, and j; n = 2 in d). Percentages represent LESR2 knockdown efficiencies after the experiments. **P

    Techniques Used: Migration, In Vitro, Allele-specific Oligonucleotide, Flow Cytometry, Staining, Wound Closure Assay, Incubation, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Derivative Assay, Expressing, Plasmid Preparation, Infection

    LESR2 regulates cell proliferation and cell migration through transcriptional regulation of KLF4 and SEMA3C. (a) Schematic representation of the experimental strategy to analyze the rescue of LESR2-ASOKD associated phenotypes with involved up- and down-regulated genes after combining LESR2 knockdown and gene targets dysregulation. (b) KLF4 expression levels after consecutive LESR2-ASOKD followed by siRNA-KD of KLF4 in neonatal LECs derived from 3 donors. (c) Quantification of the cell cycle progression analysis after 48h consecutive knockdown of LESR2 and KLF4. Statistical analysis was performed on G0 populations. (d) SEMA3C expression levels after LESR2-ASOKD in pCDH-EV and pCDH-SEMA3C infected neonatal LECs derived from 3 donors. (e) Quantification of wound closure assay (up to 9h) in neonatal LECs after the combination of SEMA3C overexpression and LESR2-ASOKD. Wound closure percentages were determined using TScratch 98 . Data are displayed as mean + SD (n = 3 in b-d; n = 10 in e). Percentages represent the knockdown efficiencies of LESR2 after the experiments. *P
    Figure Legend Snippet: LESR2 regulates cell proliferation and cell migration through transcriptional regulation of KLF4 and SEMA3C. (a) Schematic representation of the experimental strategy to analyze the rescue of LESR2-ASOKD associated phenotypes with involved up- and down-regulated genes after combining LESR2 knockdown and gene targets dysregulation. (b) KLF4 expression levels after consecutive LESR2-ASOKD followed by siRNA-KD of KLF4 in neonatal LECs derived from 3 donors. (c) Quantification of the cell cycle progression analysis after 48h consecutive knockdown of LESR2 and KLF4. Statistical analysis was performed on G0 populations. (d) SEMA3C expression levels after LESR2-ASOKD in pCDH-EV and pCDH-SEMA3C infected neonatal LECs derived from 3 donors. (e) Quantification of wound closure assay (up to 9h) in neonatal LECs after the combination of SEMA3C overexpression and LESR2-ASOKD. Wound closure percentages were determined using TScratch 98 . Data are displayed as mean + SD (n = 3 in b-d; n = 10 in e). Percentages represent the knockdown efficiencies of LESR2 after the experiments. *P

    Techniques Used: Migration, Expressing, Derivative Assay, Infection, Wound Closure Assay, Over Expression

    Transcriptional profiling after LESR2-ASOKD indicates potential functions in cell growth, cell cycle progression, and migration of LECs. (a) Schematic representation of the ASO-mediated perturbation strategy of two replicates of neonatal LECs and BECs derived from the same donor. Only samples with ASOKD efficiency > 50% were subjected to CAGE-Seq. (b) 3-dimensional scatter plot showing log2FC values calculated between single ASO against LESR2 and control ASO using EdgeR 34 . Orange and purple dots represent significantly (FDR
    Figure Legend Snippet: Transcriptional profiling after LESR2-ASOKD indicates potential functions in cell growth, cell cycle progression, and migration of LECs. (a) Schematic representation of the ASO-mediated perturbation strategy of two replicates of neonatal LECs and BECs derived from the same donor. Only samples with ASOKD efficiency > 50% were subjected to CAGE-Seq. (b) 3-dimensional scatter plot showing log2FC values calculated between single ASO against LESR2 and control ASO using EdgeR 34 . Orange and purple dots represent significantly (FDR

    Techniques Used: Migration, Allele-specific Oligonucleotide, Derivative Assay

    35) Product Images from "Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway"

    Article Title: Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

    Journal: bioRxiv

    doi: 10.1101/2020.05.03.072579

    CRISPR-Cas9 mediated TFG knock-out inhibits Wnt-dependent β-catenin stabilization and Wnt-induced reporter activity. ( A ) Schematic diagram of Cas9 cleaving the TFG genomic DNA target sequence. The target sequence (which is on the reverse strand) is shown in 5’ to 3’ orientation (left to right); the PAM (protospacer adjacent motif) sequence is in bold. ( B ) Western blot of HEK293T cells confirming complete elimination of TFG protein in Cas9 knock-out cells. β-actin was used as loading control. ( C-F ) Immunostaining for β-catenin on WT or TFG KO cells. Cells were treated with control conditioned medium or with Wnt3a conditioned medium for 3 hours before immunostaining. Note that TFG knock-out decreases Wnt3a-induced β-catenin accumulation; DAPI was used for nuclear counter-staining. Scale bars represent 20 µm. ( G ) Cas9-mediated TFG knock-out reduces response to Wnt3a by 50% in HEK293T BAR/Renilla β-catenin reporter cells. Cells were treated with Wnt3a or control medium for 16 hours before luciferase analysis. ( H ) Luciferase assay of HEK293T BAR reporter cells treated with the GSK3 inhibitor CHIR99021 at 5 µM concentration for 16 hours before being processed for luciferase assay. Note that TFG KO causes an 80% reduction of β-catenin luciferase activity in response to the GSK3 inhibition. Error bars represent standard deviation from triplicate experiments. Statistical significance was calculated with a paired 2-tailed t-Student test. * = P
    Figure Legend Snippet: CRISPR-Cas9 mediated TFG knock-out inhibits Wnt-dependent β-catenin stabilization and Wnt-induced reporter activity. ( A ) Schematic diagram of Cas9 cleaving the TFG genomic DNA target sequence. The target sequence (which is on the reverse strand) is shown in 5’ to 3’ orientation (left to right); the PAM (protospacer adjacent motif) sequence is in bold. ( B ) Western blot of HEK293T cells confirming complete elimination of TFG protein in Cas9 knock-out cells. β-actin was used as loading control. ( C-F ) Immunostaining for β-catenin on WT or TFG KO cells. Cells were treated with control conditioned medium or with Wnt3a conditioned medium for 3 hours before immunostaining. Note that TFG knock-out decreases Wnt3a-induced β-catenin accumulation; DAPI was used for nuclear counter-staining. Scale bars represent 20 µm. ( G ) Cas9-mediated TFG knock-out reduces response to Wnt3a by 50% in HEK293T BAR/Renilla β-catenin reporter cells. Cells were treated with Wnt3a or control medium for 16 hours before luciferase analysis. ( H ) Luciferase assay of HEK293T BAR reporter cells treated with the GSK3 inhibitor CHIR99021 at 5 µM concentration for 16 hours before being processed for luciferase assay. Note that TFG KO causes an 80% reduction of β-catenin luciferase activity in response to the GSK3 inhibition. Error bars represent standard deviation from triplicate experiments. Statistical significance was calculated with a paired 2-tailed t-Student test. * = P

    Techniques Used: CRISPR, Knock-Out, Activity Assay, Sequencing, Western Blot, Immunostaining, Staining, Luciferase, Concentration Assay, Inhibition, Standard Deviation

    TFG knock-out by CRISPR-Cas9 confirmed by genomic DNA sequencing. Genomic DNA was extracted from untransfected WT HEK293T cells, and from a cell clone stably transfected with a Cas9 expression vector containing an sgRNA-encoding sequence specific for human TFG. A 600 nucleotide region spanning the target sequence was amplified by PCR, cloned into sequencing vectors, and analyzed by Sanger sequencing. The reference chromatogram from sequencing results is shown below the DNA sequence. ( A ) Nucleotide sequence of TFG from wild-type (WT) HEK293 cells; the coding frame is indicated by black dots between each codon. The boxed area marks the target sequence recognized by the spacer region in the sgRNA in the WT TFG sequence. The predicted amino acid sequence is shown above the DNA sequence. ( B and C ) Sequences from a TFG knock-out clonal cell line showing the two types of mutant sequences resulting after PCR amplification of mutated genomic DNA. Each TFG allele harbored independent insertion-deletion (indel) events induced by Cas9 as a result of non-homologous end joining (NHEJ) repair. In Mutation 1 (Mut1) Cas9 induced a deletion of a short nucleotide sequence (8 nucleotides, AAGACCCC). Mutation 2 (Mut 2) resulted in the insertion of an extra adenine between the original CCA and AGA codons. Both indel events induced a frameshift mutation, introducing an early STOP codon indicated by an asterisk, generating truncated TFG mutant proteins of approximately 90 amino acids.
    Figure Legend Snippet: TFG knock-out by CRISPR-Cas9 confirmed by genomic DNA sequencing. Genomic DNA was extracted from untransfected WT HEK293T cells, and from a cell clone stably transfected with a Cas9 expression vector containing an sgRNA-encoding sequence specific for human TFG. A 600 nucleotide region spanning the target sequence was amplified by PCR, cloned into sequencing vectors, and analyzed by Sanger sequencing. The reference chromatogram from sequencing results is shown below the DNA sequence. ( A ) Nucleotide sequence of TFG from wild-type (WT) HEK293 cells; the coding frame is indicated by black dots between each codon. The boxed area marks the target sequence recognized by the spacer region in the sgRNA in the WT TFG sequence. The predicted amino acid sequence is shown above the DNA sequence. ( B and C ) Sequences from a TFG knock-out clonal cell line showing the two types of mutant sequences resulting after PCR amplification of mutated genomic DNA. Each TFG allele harbored independent insertion-deletion (indel) events induced by Cas9 as a result of non-homologous end joining (NHEJ) repair. In Mutation 1 (Mut1) Cas9 induced a deletion of a short nucleotide sequence (8 nucleotides, AAGACCCC). Mutation 2 (Mut 2) resulted in the insertion of an extra adenine between the original CCA and AGA codons. Both indel events induced a frameshift mutation, introducing an early STOP codon indicated by an asterisk, generating truncated TFG mutant proteins of approximately 90 amino acids.

    Techniques Used: Knock-Out, CRISPR, DNA Sequencing, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay, Mutagenesis, Non-Homologous End Joining

    36) Product Images from "Evolutionary Changes in Left-Right Visceral Asymmetry in Astyanax Cavefish"

    Article Title: Evolutionary Changes in Left-Right Visceral Asymmetry in Astyanax Cavefish

    Journal: bioRxiv

    doi: 10.1101/2020.05.15.098483

    Maternal genetic effects on heart looping laterality. A. Reciprocal crosses between surface fish and cavefish gametes in both directions were used to assay for maternal genetic effects. B. Bar graphs showing the percentage of heart looping types in the F1 progeny of F61 cavefish x cavefish (top row), F61 cavefish female x F29 male (second from top row), F29 surface female x F61 cavefish male (second from bottom row), and F29 surface fish x F29 surface fish male (bottom row) crosses shown from top to bottom. Number of larvae analyzed are indicated at the right of each bar in B.
    Figure Legend Snippet: Maternal genetic effects on heart looping laterality. A. Reciprocal crosses between surface fish and cavefish gametes in both directions were used to assay for maternal genetic effects. B. Bar graphs showing the percentage of heart looping types in the F1 progeny of F61 cavefish x cavefish (top row), F61 cavefish female x F29 male (second from top row), F29 surface female x F61 cavefish male (second from bottom row), and F29 surface fish x F29 surface fish male (bottom row) crosses shown from top to bottom. Number of larvae analyzed are indicated at the right of each bar in B.

    Techniques Used: Fluorescence In Situ Hybridization

    37) Product Images from "A comprehensive assay for targeted multiplex amplification of human DNA sequences"

    Article Title: A comprehensive assay for targeted multiplex amplification of human DNA sequences

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

    doi: 10.1073/pnas.0803240105

    The median CEL intensities for each amplicon obtained by using Stoffel DNA polymerase and Phusion DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were
    Figure Legend Snippet: The median CEL intensities for each amplicon obtained by using Stoffel DNA polymerase and Phusion DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were

    Techniques Used: Amplification

    38) Product Images from "Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases"

    Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases

    Journal: Nucleic Acid Therapeutics

    doi: 10.1089/nat.2014.0513

    Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.
    Figure Legend Snippet: Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Amplification, Modification

    39) Product Images from "Resistance to Ceftazidime/Avibactam Plus Meropenem/Vaborbactam When Both are Used Together Achieved in Four Steps From Metallo-β-Lactamase Negative Klebsiella pneumoniae"

    Article Title: Resistance to Ceftazidime/Avibactam Plus Meropenem/Vaborbactam When Both are Used Together Achieved in Four Steps From Metallo-β-Lactamase Negative Klebsiella pneumoniae

    Journal: bioRxiv

    doi: 10.1101/2020.03.09.983304

    Checkerboard assays for ceftazidime and meropenem in the presence of avibactam and vaborbactam. Each image represents duplicate assays for an 8×8 array of wells in a 96-well plate. All wells contained CA-MHB including avibactam (4 μg.mL −1 ) and vaborbactam (8 μg.mL −1 ). A serial dilution of meropenem (MEM, x-axis) and ceftazidime (CAZ, y-axis) was created from 32 μg.mL −1 in each plate as recorded. All wells were inoculated with a suspension of bacteria, made as per CLSI microtiter MIC guidelines ( 33 ), and the plate was incubated at 37°C for 20h. Growth was recorded by measuring OD 600 and growth above background (broth) is recorded as a yellow block. Growth at 8 μg.mL −1 ceftazidime and 8 μg.mL −1 meropenem (this position indicated in red) in the presence of vaborbactam and avibactam defines resistance based on CLSI breakpoints ( 34 ). Bacterial suspensions used were: for images in the top row, KP21[ ramR ] ompK36 ; second row, KP21[ ramR ] ompK36 (pOXA-232); third row, KP21[ ramR ] M[ ompK36 ](pOXA-232); fourth row, KP21[ ramR ] M[ ompK36 ] ompK35 (pOXA-232); fifth row, KP47 ompK36 (pOXA-232). In each case, bacteria also carry the following plasmids (where tested): images in first column, pCTX-M-14 P170S; second column, pKPC-3; third column, pKPC-3-D178Y; fourth column, pKPC-3-V239G.
    Figure Legend Snippet: Checkerboard assays for ceftazidime and meropenem in the presence of avibactam and vaborbactam. Each image represents duplicate assays for an 8×8 array of wells in a 96-well plate. All wells contained CA-MHB including avibactam (4 μg.mL −1 ) and vaborbactam (8 μg.mL −1 ). A serial dilution of meropenem (MEM, x-axis) and ceftazidime (CAZ, y-axis) was created from 32 μg.mL −1 in each plate as recorded. All wells were inoculated with a suspension of bacteria, made as per CLSI microtiter MIC guidelines ( 33 ), and the plate was incubated at 37°C for 20h. Growth was recorded by measuring OD 600 and growth above background (broth) is recorded as a yellow block. Growth at 8 μg.mL −1 ceftazidime and 8 μg.mL −1 meropenem (this position indicated in red) in the presence of vaborbactam and avibactam defines resistance based on CLSI breakpoints ( 34 ). Bacterial suspensions used were: for images in the top row, KP21[ ramR ] ompK36 ; second row, KP21[ ramR ] ompK36 (pOXA-232); third row, KP21[ ramR ] M[ ompK36 ](pOXA-232); fourth row, KP21[ ramR ] M[ ompK36 ] ompK35 (pOXA-232); fifth row, KP47 ompK36 (pOXA-232). In each case, bacteria also carry the following plasmids (where tested): images in first column, pCTX-M-14 P170S; second column, pKPC-3; third column, pKPC-3-D178Y; fourth column, pKPC-3-V239G.

    Techniques Used: Serial Dilution, Incubation, Blocking Assay

    40) Product Images from "Cancer modeling in colorectal organoids reveals intrinsic differences between oncogenic RAS and BRAF variants"

    Article Title: Cancer modeling in colorectal organoids reveals intrinsic differences between oncogenic RAS and BRAF variants

    Journal: bioRxiv

    doi: 10.1101/860122

    Generation of oncogenic BRAF and RAS knock-in variants in CRC PDOs. ( A ) Genetic strategy to target KRAS, NRAS and BRAF locus for homologous directed repair using CRISPR/Cas9 technology. Red and blue lines indicate oncogenic missense and silent mutations, respectively. Black boxes illustrate exons, separated by introns. Red scissor shows sgRNA-generated double stranded break. Black arrows illustrate PCR primer pairs that were used for the identification of knock-in clones. ( B ) Puromycin selection strategy to generate RAS and BRAF mutant CRC PDOs after CRISPR-mediated homologous recombination. ( C ) Per mutation the agarose electrophoresis gels showing the ∼1kb PCR product of the knock-in allele in 3 monoclonal lines per mutations. Sanger sequencing confirms presence of both knock-in and WT alleles. DNA sequences show introduction of missense (red) and silent (blue) mutations. The mRNA expression levels of wild-type and mutant alleles was analyzed using qPCR. The relative expression of each allele was normalized to the B2M housekeeping gene (representative from n = 3 independent experiments). ( D ) Western blot analysis shows enhanced RAS activity (GTP-loading) in RAS mutant CRC PDOs compared to P18T organoids. KRAS and NRAS immunoblots from RAS pull-down assays (RAS-GTP) and total lysates (loading control) are shown for KRAS and NRAS mutant organoids, respectively. Representative from n=2 independent experiments.
    Figure Legend Snippet: Generation of oncogenic BRAF and RAS knock-in variants in CRC PDOs. ( A ) Genetic strategy to target KRAS, NRAS and BRAF locus for homologous directed repair using CRISPR/Cas9 technology. Red and blue lines indicate oncogenic missense and silent mutations, respectively. Black boxes illustrate exons, separated by introns. Red scissor shows sgRNA-generated double stranded break. Black arrows illustrate PCR primer pairs that were used for the identification of knock-in clones. ( B ) Puromycin selection strategy to generate RAS and BRAF mutant CRC PDOs after CRISPR-mediated homologous recombination. ( C ) Per mutation the agarose electrophoresis gels showing the ∼1kb PCR product of the knock-in allele in 3 monoclonal lines per mutations. Sanger sequencing confirms presence of both knock-in and WT alleles. DNA sequences show introduction of missense (red) and silent (blue) mutations. The mRNA expression levels of wild-type and mutant alleles was analyzed using qPCR. The relative expression of each allele was normalized to the B2M housekeeping gene (representative from n = 3 independent experiments). ( D ) Western blot analysis shows enhanced RAS activity (GTP-loading) in RAS mutant CRC PDOs compared to P18T organoids. KRAS and NRAS immunoblots from RAS pull-down assays (RAS-GTP) and total lysates (loading control) are shown for KRAS and NRAS mutant organoids, respectively. Representative from n=2 independent experiments.

    Techniques Used: Knock-In, CRISPR, Generated, Polymerase Chain Reaction, Clone Assay, Selection, Mutagenesis, Homologous Recombination, Electrophoresis, Sequencing, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay

    Karyotype and gene expression profiles of RPM lines. ( A ) DNA copy number alterations of monoclonal RPM lines (clones # 1-3) and parental (P) P18T organoids. Centromeric regions (thin black line in chromosomes) are excluded due to repetitive nature of DNA sequence. Dashed lines indicate the genetic location of BRAF, KRAS and NRAS loci . Colors per (sub)chromosome indicate CNA according to legend. ( B ) Principle Component Analysis of gene expression levels for monoclonal RPM lines and parental P18T organoids (bulk and monoclonal lines # 1-3) during unperturbed growth conditions (DMSO) and pan-HER inhibition (1 µM afatinib for 24 hr). Left panel indicates treatment. Right panel indicates clone identities. Hue at background corresponds to treatment. ( C ) Heatmap of significantly downregulated genes (p -adjusted
    Figure Legend Snippet: Karyotype and gene expression profiles of RPM lines. ( A ) DNA copy number alterations of monoclonal RPM lines (clones # 1-3) and parental (P) P18T organoids. Centromeric regions (thin black line in chromosomes) are excluded due to repetitive nature of DNA sequence. Dashed lines indicate the genetic location of BRAF, KRAS and NRAS loci . Colors per (sub)chromosome indicate CNA according to legend. ( B ) Principle Component Analysis of gene expression levels for monoclonal RPM lines and parental P18T organoids (bulk and monoclonal lines # 1-3) during unperturbed growth conditions (DMSO) and pan-HER inhibition (1 µM afatinib for 24 hr). Left panel indicates treatment. Right panel indicates clone identities. Hue at background corresponds to treatment. ( C ) Heatmap of significantly downregulated genes (p -adjusted

    Techniques Used: Expressing, Sequencing, Inhibition

    KRAS G12D and BRAF V600E knock-in mutations promote organoid survival and growth. ( A ) Quantitative analysis of organoid size and number in isogenic RPM lines during normal culture conditions (DMSO) at different time points after organoid plating. Size and number of viable organoids were measured by uptake of fluorescent calcein green (see methods). Each dot represents one organoid 76 . Data from 1 8-well Lab-Tek chambered coverglass is shown. ( B ) Representative fluorescent pictures of parental P18T, or KRAS G12D , KRAS G13D , NRAS G12D and BRAF V600E knock-in organoids (clone # 1) after 7 days of DMSO or afatinib treatment (1 μM). Scale bars, 100 μM. Hoechst (blue) and DRAQ7 (red) was used to visualize nuclei and dead cells, respectively. ( C ) Representative bright field pictures of parental P18T, or KRAS G12D , KRAS G13D , NRAS G12D and BRAF V600E knock-in organoids (clone # 1) 7 days after release of afatinib treatment (day 14). Representative zoom-in panels show fluorescent calcein green signal in living cells. Asterisks indicate autofluorescence of dead material ( Suppl. Fig 3C ). ( D ) Quantitative analysis of organoid growth and viability in isogenic RPM lines prior (day 0), during (day 7) and after (day 14) treatment with afatinib (1 μM). Size and number of viable organoids were measured by uptake of fluorescent calcein green (see methods). Each dot represents one organoid 76 . Data from 1 8-well Lab-Tek chambered coverglass is shown.
    Figure Legend Snippet: KRAS G12D and BRAF V600E knock-in mutations promote organoid survival and growth. ( A ) Quantitative analysis of organoid size and number in isogenic RPM lines during normal culture conditions (DMSO) at different time points after organoid plating. Size and number of viable organoids were measured by uptake of fluorescent calcein green (see methods). Each dot represents one organoid 76 . Data from 1 8-well Lab-Tek chambered coverglass is shown. ( B ) Representative fluorescent pictures of parental P18T, or KRAS G12D , KRAS G13D , NRAS G12D and BRAF V600E knock-in organoids (clone # 1) after 7 days of DMSO or afatinib treatment (1 μM). Scale bars, 100 μM. Hoechst (blue) and DRAQ7 (red) was used to visualize nuclei and dead cells, respectively. ( C ) Representative bright field pictures of parental P18T, or KRAS G12D , KRAS G13D , NRAS G12D and BRAF V600E knock-in organoids (clone # 1) 7 days after release of afatinib treatment (day 14). Representative zoom-in panels show fluorescent calcein green signal in living cells. Asterisks indicate autofluorescence of dead material ( Suppl. Fig 3C ). ( D ) Quantitative analysis of organoid growth and viability in isogenic RPM lines prior (day 0), during (day 7) and after (day 14) treatment with afatinib (1 μM). Size and number of viable organoids were measured by uptake of fluorescent calcein green (see methods). Each dot represents one organoid 76 . Data from 1 8-well Lab-Tek chambered coverglass is shown.

    Techniques Used: Knock-In

    KRAS G12D and BRAF V600E RPM lines show residual MAPK pathway activity in the presence of pan-HER inhibition. ( A ) Organoids expressing oncogenic KRAS (G12D and G13D), NRAS (G12D) and BRAF (V600E) variants show enhanced basal ERK phosphorylation levels compared to P18T organoids. Pan-HER inhibition (1 µM afatinib) shows sustained ERK and MEK phosphorylation in KRAS G12D and BRAF V600E organoids compared to P18T, KRAS G13D and NRAS G12D organoids. Top panels are representative biochemistry experiments on clone # 1 from n=3. Bottom scatter plot depicts ERK phosphorylation levels normalized to GAPDH for all clones (n≥4). Baseline of P18T (DMSO) is set at 1. ( B ) Top panels depict biochemistry on RAS activity (GTP-loading) in unperturbed culture conditions and pan-HER inhibition (1 µM afatinib) for KRAS (G12D and G13D) and NRAS (G12D) mutant clones # 1 and 2 compared to P18T CRC organoids. RAS immunoblots from RAS pull-down assay are shown (RAS-GTP), together with a RAS immunoblot from total cell lysates as loading control. HRAS, KRAS, and NRAS isoforms are detected in mutant KRAS pull-down assays. NRAS isoforms are detected in mutant NRAS pull-down assays. Representative from n = 3 independent experiments. Scatter plots below depict RAS-GTP levels normalized to GAPDH for all clones (n≥3). Baseline of P18T (DMSO) is set at 1.
    Figure Legend Snippet: KRAS G12D and BRAF V600E RPM lines show residual MAPK pathway activity in the presence of pan-HER inhibition. ( A ) Organoids expressing oncogenic KRAS (G12D and G13D), NRAS (G12D) and BRAF (V600E) variants show enhanced basal ERK phosphorylation levels compared to P18T organoids. Pan-HER inhibition (1 µM afatinib) shows sustained ERK and MEK phosphorylation in KRAS G12D and BRAF V600E organoids compared to P18T, KRAS G13D and NRAS G12D organoids. Top panels are representative biochemistry experiments on clone # 1 from n=3. Bottom scatter plot depicts ERK phosphorylation levels normalized to GAPDH for all clones (n≥4). Baseline of P18T (DMSO) is set at 1. ( B ) Top panels depict biochemistry on RAS activity (GTP-loading) in unperturbed culture conditions and pan-HER inhibition (1 µM afatinib) for KRAS (G12D and G13D) and NRAS (G12D) mutant clones # 1 and 2 compared to P18T CRC organoids. RAS immunoblots from RAS pull-down assay are shown (RAS-GTP), together with a RAS immunoblot from total cell lysates as loading control. HRAS, KRAS, and NRAS isoforms are detected in mutant KRAS pull-down assays. NRAS isoforms are detected in mutant NRAS pull-down assays. Representative from n = 3 independent experiments. Scatter plots below depict RAS-GTP levels normalized to GAPDH for all clones (n≥3). Baseline of P18T (DMSO) is set at 1.

    Techniques Used: Activity Assay, Inhibition, Expressing, Clone Assay, Mutagenesis, Western Blot, Pull Down Assay

    Differential drug sensitivities of oncogenic RAS and BRAF knock-in CRC PDOs to targeted MAPK pathway inhibition. ( A ) Schematic overview of the drug screening method. In short, a 7-day drug screen was initiated on 5 days old P18T and RPM organoids expressing different RAS and BRAF mutations. ( B - H ) Heat maps of dose-response measurements (cell viability) in CRC PDO lines to ( B ) afatinib, ( C ) selumetinib, ( D ) afatinib plus selumetinib, ( E ) SCH772984, ( F ) SCH772984 plus selumetinib, ( G ) vemurafenib and ( H ) vemurafenib plus afatinib. Organoids were treated (7 days) with vehicle (DMSO) or inhibitors targeting the EGFR-RAS-ERK pathway (5 nM – 30 μM range, in 14 logarithmic intervals). Red represents maximal cell death and green represents maximal viability. Drug names and their nominal targets are indicated above and the MAPK pathway mutant status per line at the left. Average of 2 technical replicates.
    Figure Legend Snippet: Differential drug sensitivities of oncogenic RAS and BRAF knock-in CRC PDOs to targeted MAPK pathway inhibition. ( A ) Schematic overview of the drug screening method. In short, a 7-day drug screen was initiated on 5 days old P18T and RPM organoids expressing different RAS and BRAF mutations. ( B - H ) Heat maps of dose-response measurements (cell viability) in CRC PDO lines to ( B ) afatinib, ( C ) selumetinib, ( D ) afatinib plus selumetinib, ( E ) SCH772984, ( F ) SCH772984 plus selumetinib, ( G ) vemurafenib and ( H ) vemurafenib plus afatinib. Organoids were treated (7 days) with vehicle (DMSO) or inhibitors targeting the EGFR-RAS-ERK pathway (5 nM – 30 μM range, in 14 logarithmic intervals). Red represents maximal cell death and green represents maximal viability. Drug names and their nominal targets are indicated above and the MAPK pathway mutant status per line at the left. Average of 2 technical replicates.

    Techniques Used: Knock-In, Inhibition, Expressing, Mutagenesis

    Related Articles

    Sequencing:

    Article Title: Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses
    Article Snippet: .. This result demonstrated that adequate distribution of sequencing reads between segments were obtained from a DNA library prepared from samples amplified by Phusion DNA polymerase (DNA library), and by whole-RNA library. .. Consistency of Illumina sequencing, and sequencing analysis of A/PR/8/34 and A/California/07/2009 strains Center for Biologics Evaluation and Research (CBER) stock of A/PR/8/34 was kindly provided by Dr. Peter Palese at Mount.

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
    Article Snippet: .. Such changes were still observed after amplification with Phusion High Fidelity DNA Polymerase, with 3/15 (20%) of single allele clones obtained from HepG2 and Huh-7 DNA showing apparent poly(T) slippage and also four sequence variants observed that were not seen in other clones of the same haplotype ( ). .. Overall, despite this sequence heterogeneity, the polymorphisms confirmed two different haplotypes in HepG2 DNA, consistent with the presence of two alleles of the pre-mir-122 stem-loop region.

    Clone Assay:

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
    Article Snippet: .. Such changes were still observed after amplification with Phusion High Fidelity DNA Polymerase, with 3/15 (20%) of single allele clones obtained from HepG2 and Huh-7 DNA showing apparent poly(T) slippage and also four sequence variants observed that were not seen in other clones of the same haplotype ( ). .. Overall, despite this sequence heterogeneity, the polymorphisms confirmed two different haplotypes in HepG2 DNA, consistent with the presence of two alleles of the pre-mir-122 stem-loop region.

    Amplification:

    Article Title: Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses
    Article Snippet: .. This result demonstrated that adequate distribution of sequencing reads between segments were obtained from a DNA library prepared from samples amplified by Phusion DNA polymerase (DNA library), and by whole-RNA library. .. Consistency of Illumina sequencing, and sequencing analysis of A/PR/8/34 and A/California/07/2009 strains Center for Biologics Evaluation and Research (CBER) stock of A/PR/8/34 was kindly provided by Dr. Peter Palese at Mount.

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
    Article Snippet: .. Such changes were still observed after amplification with Phusion High Fidelity DNA Polymerase, with 3/15 (20%) of single allele clones obtained from HepG2 and Huh-7 DNA showing apparent poly(T) slippage and also four sequence variants observed that were not seen in other clones of the same haplotype ( ). .. Overall, despite this sequence heterogeneity, the polymorphisms confirmed two different haplotypes in HepG2 DNA, consistent with the presence of two alleles of the pre-mir-122 stem-loop region.

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction
    Article Snippet: .. Successful PCR amplification has also been obtained with a Phusion High‐Fidelity DNA polymerase (New England Biolabs; M0530; see Fig. A). .. This polymerase was tested in the following PCR conditions for PCR products of ∼1.2 kb: 1 cycle at 98°C for 30 s followed by 35 cycles of 98°C for 10 s, 58°C for 20 s, 72°C for 45 s, and finally 1 cycle of 72°C for 5 min. 8 Following the PCR, mix 5 µl of the PCR reaction with 1 µl of 6× DNA loading dye and load the reactions on an agarose gel (Voytas, ).

    DNA Purification:

    Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases
    Article Snippet: .. Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. .. Synthetic oligodeoxynucleotides, including a target ( X ) and a probe ( P ), were synthesized by Integrated DNA Technologies, Inc. and purified by high-performance liquid chromatography (HPLC).

    Polymerase Chain Reaction:

    Article Title: Variations of five eIF4E genes across cassava accessions exhibiting tolerant and susceptible responses to cassava brown streak disease
    Article Snippet: .. PCR was performed in a 20 μl reaction volume containing 10 unit Phusion DNA polymerase (NEB, Ipswich, MA), 1 μl of 1:5 diluted cDNA template, 1X Phusion PCR buffer, 5 μM each of upstream and downstream primers, and 250 nM dNTP with the following cycling condition: 98°C for 1 minute; 35 cycles of 98°C for 15 seconds, 56°C for 15 seconds, and 72°C for 45 seconds; and finally 72°C for 5 minutes. .. Primers were designed according to five annotated eIF4E transcripts identified in the draft cassava genomic sequence (Manihot esculenta v4.1) published in Phytozome ( http://phytozome.jgi.doe.gov ) in 2013, prior to the availability of the current cassava genome V6.1 ( ).

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction
    Article Snippet: .. Successful PCR amplification has also been obtained with a Phusion High‐Fidelity DNA polymerase (New England Biolabs; M0530; see Fig. A). .. This polymerase was tested in the following PCR conditions for PCR products of ∼1.2 kb: 1 cycle at 98°C for 30 s followed by 35 cycles of 98°C for 10 s, 58°C for 20 s, 72°C for 45 s, and finally 1 cycle of 72°C for 5 min. 8 Following the PCR, mix 5 µl of the PCR reaction with 1 µl of 6× DNA loading dye and load the reactions on an agarose gel (Voytas, ).

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    New England Biolabs phusion high fidelity dna polymerase
    The median CEL intensities for each amplicon obtained by using Stoffel <t>DNA</t> polymerase and <t>Phusion</t> DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The median CEL intensities for each amplicon obtained by using Stoffel DNA polymerase and Phusion DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were

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

    Article Title: A comprehensive assay for targeted multiplex amplification of human DNA sequences

    doi: 10.1073/pnas.0803240105

    Figure Lengend Snippet: The median CEL intensities for each amplicon obtained by using Stoffel DNA polymerase and Phusion DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were

    Article Snippet: The extension was performed by addition of 0.4 units of Phusion High-Fidelity DNA Polymerase (New England Biolabs), 3 μl 1.0 mM dNTP, 5 units Ampligase (Epicenter Biotechnologies) in a 15-μl volume at 60°C for 15 min followed by 72°C for 15 min.

    Techniques: Amplification

    Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Journal: PLoS ONE

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells

    doi: 10.1371/journal.pone.0122471

    Figure Lengend Snippet: Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Article Snippet: Such changes were still observed after amplification with Phusion High Fidelity DNA Polymerase, with 3/15 (20%) of single allele clones obtained from HepG2 and Huh-7 DNA showing apparent poly(T) slippage and also four sequence variants observed that were not seen in other clones of the same haplotype ( ).

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

    PAGE electrophoresis of PEAR products. For dNTPs, lowercase letters (agct) represents natural dNTPs, and uppercase letters (AGCT) represents dNTPαSs. (A) PEAR products incorporating natural or dATPαS, dGTPαS, dCTPαS, dTTPαS: Lane 1: natural dNTPs; Lane 2: dATPαSs; Lane 3: No PspGI control; Lane 4: No Phusion DNA polymerase control; Lane 5: No dATP control; Lane 6∶10bp DNA ladder; Lane 7: dGTPαS; Lane 8: No PspGI control; Lane 9: No Phusion DNA polymerase control; Lane 10: No dCTP control; Lane 11: dCTPαSs; Lane 12: No PspGI control; Lane 13: No Phusion DNA polymerase control; Lane 14: No dCTP control; Lane 15: dTTPαSs; Lane 16: No PspGI control; Lane 17: No Phusion DNA polymerase control; Lane 18: No dTTP control; Lane 19∶10bp DNA ladder. (B) PEAR products incorporating one or two kind of dNTPαSs: Lane 1: natural dNTPs; Lane 2–5: one kind of dNTPαSs; Lane 6–8: two kind of dNTPαSs; Lane 9: No dNTPs control; Lane 10∶10bp DNA ladder; (C) Full digestion of PEAR products incorporating different dNTPs or dNTPαSs.

    Journal: PLoS ONE

    Article Title: Preparation of 5?-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)

    doi: 10.1371/journal.pone.0067558

    Figure Lengend Snippet: PAGE electrophoresis of PEAR products. For dNTPs, lowercase letters (agct) represents natural dNTPs, and uppercase letters (AGCT) represents dNTPαSs. (A) PEAR products incorporating natural or dATPαS, dGTPαS, dCTPαS, dTTPαS: Lane 1: natural dNTPs; Lane 2: dATPαSs; Lane 3: No PspGI control; Lane 4: No Phusion DNA polymerase control; Lane 5: No dATP control; Lane 6∶10bp DNA ladder; Lane 7: dGTPαS; Lane 8: No PspGI control; Lane 9: No Phusion DNA polymerase control; Lane 10: No dCTP control; Lane 11: dCTPαSs; Lane 12: No PspGI control; Lane 13: No Phusion DNA polymerase control; Lane 14: No dCTP control; Lane 15: dTTPαSs; Lane 16: No PspGI control; Lane 17: No Phusion DNA polymerase control; Lane 18: No dTTP control; Lane 19∶10bp DNA ladder. (B) PEAR products incorporating one or two kind of dNTPαSs: Lane 1: natural dNTPs; Lane 2–5: one kind of dNTPαSs; Lane 6–8: two kind of dNTPαSs; Lane 9: No dNTPs control; Lane 10∶10bp DNA ladder; (C) Full digestion of PEAR products incorporating different dNTPs or dNTPαSs.

    Article Snippet: Materials Phusion high fidelity DNA polymerase, highly thermostable restriction enzyme PspGI and dNTPs are purchased from New England Biolabs , Inc .

    Techniques: Polyacrylamide Gel Electrophoresis, Electrophoresis

    PCR to test the efficiency of polymerases in amplifying PCR products from supernatants from different spore concentrations of the A. fumigatus wild‐type strain with primers ITS1/D2 (expected PCR band sizes is ∼1.2 kb). ( A ) Phusion High‐Fidelity DNA polymerase (New England Biolabs). ( B ) MyTaq RED Mix DNA polymerase (Bioline). P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).

    Journal: Current Protocols in Microbiology

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction

    doi: 10.1002/cpmc.89

    Figure Lengend Snippet: PCR to test the efficiency of polymerases in amplifying PCR products from supernatants from different spore concentrations of the A. fumigatus wild‐type strain with primers ITS1/D2 (expected PCR band sizes is ∼1.2 kb). ( A ) Phusion High‐Fidelity DNA polymerase (New England Biolabs). ( B ) MyTaq RED Mix DNA polymerase (Bioline). P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).

    Article Snippet: Successful PCR amplification has also been obtained with a Phusion High‐Fidelity DNA polymerase (New England Biolabs; M0530; see Fig. A).

    Techniques: Polymerase Chain Reaction, Amplification, Negative Control