Structured Review

Bio-Rad transgenes
Schematic diagrams illustrating in vivo rearrangements of λgt10- lacZ <t>transgene.</t> Panels ( A ) through ( E ) show examples of rearranged λgt10- lacZ copies (R1–R5) that correspond to the rearrangement fragments of
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1) Product Images from "Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements"

Article Title: Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements

Journal: Mutagenesis

doi: 10.1093/mutage/geq048

Schematic diagrams illustrating in vivo rearrangements of λgt10- lacZ transgene. Panels ( A ) through ( E ) show examples of rearranged λgt10- lacZ copies (R1–R5) that correspond to the rearrangement fragments of
Figure Legend Snippet: Schematic diagrams illustrating in vivo rearrangements of λgt10- lacZ transgene. Panels ( A ) through ( E ) show examples of rearranged λgt10- lacZ copies (R1–R5) that correspond to the rearrangement fragments of

Techniques Used: In Vivo

Summary of RT–PCR analysis of in vivo transgene copy number. Standard curves were based on quantifications of the ori amplicons, using replicated sets of template composed of CD2F 1 non-transgenic mouse DNA mixed with λgt10- lacZ (see Figure 3 Materials and methods) to generate haploid standards of 1, 2.5, 5, 10, 20, 40 and 50 copies. Cycle thresholds ( C t ) and efficiency parameters were obtained by iCycler software and show a linear correlation with log copy number values ( R 2 = 0.9924). Shown are average C t values for: ori standards (open circles), single-copy annexin V exon 4 (closed triangle, A); R5 rearrangement (closed circle, R5); interpolated copies of Muta™mouse ori (closed diamond, MM) and 18S (closed box, 18S).
Figure Legend Snippet: Summary of RT–PCR analysis of in vivo transgene copy number. Standard curves were based on quantifications of the ori amplicons, using replicated sets of template composed of CD2F 1 non-transgenic mouse DNA mixed with λgt10- lacZ (see Figure 3 Materials and methods) to generate haploid standards of 1, 2.5, 5, 10, 20, 40 and 50 copies. Cycle thresholds ( C t ) and efficiency parameters were obtained by iCycler software and show a linear correlation with log copy number values ( R 2 = 0.9924). Shown are average C t values for: ori standards (open circles), single-copy annexin V exon 4 (closed triangle, A); R5 rearrangement (closed circle, R5); interpolated copies of Muta™mouse ori (closed diamond, MM) and 18S (closed box, 18S).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vivo, Transgenic Assay, Software

Model of the Muta™Mouse λgt10- lacZ transgene derived by sequence analysis. The transgene monomer has 47 513 bp and 57 ORFs and is based on nucleotide sequencing of PCR amplicons derived from systematic scanning of functional regions of λgt10- lacZ using Muta™Mouse genomic DNA from tissues of both gender and λgt10- lacZ in vivo copies rescued by in vitro phage packaging and commercial stocks of λCI857 and λgt10. Data include the b527 deletion and unfinished parts of bacteriophage imm434 (accession numbers M60848, Y00118) and the 5.2 kb EcoR1-DraI fragment of pMC1511, which contains the E.coli lacZ mutation reporter (GenBank® L08935) (hatched-box). The GenBank® accession numbers for sequences other than those reported already for λ (NC_001416; 48.5 kb) are given in Table II . Functional regions included left (COS L ) and right (COS R ) cohesive ends and ORFs required for virion assembly and DNA packaging and origin of λ DNA replication (ORI). Arrows show orientation of ORFs common to the 48.5 kb λ bacteriophage as identified by λ gene nomenclature (see NC_001416). Novel in vivo copy rearrangements are described in Figure 2 and Table II . The large open box spans a novel finding of a region of substitution by lambdoid Rac prophage tail fiber assembly gene ( lom and ORFs 401, 314 and 194) found also in λgt10 and λCI857 commercial stocks. Also identified are crossover hot spot instigator motifs (Chi sites) conveying potential for lacZ recombination with the E.coli genome. The symbol χ + signifies a fully functional Chi motif (starts at nucleotide position 3508 on the reverse or antistrand with respect to E.coli lacZ (accession J01636). χ 0 signifies a one base difference version of the Chi motif that requires a single mutation for full function. These χ 0 sites start at nucleotide positions 3149 and 3152 for the two on the anti-strand and position 3248 for the one on the sense strand. MAR-1 and MAR-2 show location of sequences identified as matrix-associated regions using a computational approach ( 15 ). MAR-1 is located 5′ to the lambda INT gene and MAR-2 is located in the 3′ non-coding λgt-10 region and they represent regions that may contribute to higher order mammalian chromosome loop structures. In a head-to-tail arrangement of transgenes, MAR-2 would be located within 1.4 kb of the COS L site and in the vicinity of rearrangements featured in Figure 2 .
Figure Legend Snippet: Model of the Muta™Mouse λgt10- lacZ transgene derived by sequence analysis. The transgene monomer has 47 513 bp and 57 ORFs and is based on nucleotide sequencing of PCR amplicons derived from systematic scanning of functional regions of λgt10- lacZ using Muta™Mouse genomic DNA from tissues of both gender and λgt10- lacZ in vivo copies rescued by in vitro phage packaging and commercial stocks of λCI857 and λgt10. Data include the b527 deletion and unfinished parts of bacteriophage imm434 (accession numbers M60848, Y00118) and the 5.2 kb EcoR1-DraI fragment of pMC1511, which contains the E.coli lacZ mutation reporter (GenBank® L08935) (hatched-box). The GenBank® accession numbers for sequences other than those reported already for λ (NC_001416; 48.5 kb) are given in Table II . Functional regions included left (COS L ) and right (COS R ) cohesive ends and ORFs required for virion assembly and DNA packaging and origin of λ DNA replication (ORI). Arrows show orientation of ORFs common to the 48.5 kb λ bacteriophage as identified by λ gene nomenclature (see NC_001416). Novel in vivo copy rearrangements are described in Figure 2 and Table II . The large open box spans a novel finding of a region of substitution by lambdoid Rac prophage tail fiber assembly gene ( lom and ORFs 401, 314 and 194) found also in λgt10 and λCI857 commercial stocks. Also identified are crossover hot spot instigator motifs (Chi sites) conveying potential for lacZ recombination with the E.coli genome. The symbol χ + signifies a fully functional Chi motif (starts at nucleotide position 3508 on the reverse or antistrand with respect to E.coli lacZ (accession J01636). χ 0 signifies a one base difference version of the Chi motif that requires a single mutation for full function. These χ 0 sites start at nucleotide positions 3149 and 3152 for the two on the anti-strand and position 3248 for the one on the sense strand. MAR-1 and MAR-2 show location of sequences identified as matrix-associated regions using a computational approach ( 15 ). MAR-1 is located 5′ to the lambda INT gene and MAR-2 is located in the 3′ non-coding λgt-10 region and they represent regions that may contribute to higher order mammalian chromosome loop structures. In a head-to-tail arrangement of transgenes, MAR-2 would be located within 1.4 kb of the COS L site and in the vicinity of rearrangements featured in Figure 2 .

Techniques Used: Derivative Assay, Sequencing, Polymerase Chain Reaction, Functional Assay, In Vivo, In Vitro, Mutagenesis

2) Product Images from "Splicing Factor 3B Subunit 1 Interacts with HIV Tat and Plays a Role in Viral Transcription and Reactivation from Latency"

Article Title: Splicing Factor 3B Subunit 1 Interacts with HIV Tat and Plays a Role in Viral Transcription and Reactivation from Latency

Journal: mBio

doi: 10.1128/mBio.01423-18

Inhibition of SF3B1 reduces HIV products. (A) U87/CD4/CXCR4 cells were infected with replication-competent HIV-1 for 24 h. Total cellular RNA was obtained, and qRT-PCR was performed for the unspliced (US), singly spliced (SS), and multiply spliced (MS) forms of HIV using specific primers. RNA fold change was calculated relative to DMSO treatment. (B) 293T cells were infected with HIV Δ env for 24 h in the presence of DMSO or sudemycin D6. Western blots were performed on cell lysates for HIV Gag products. Three experiments were quantitated (C). (D) 293T cells were infected with HIV Δ env for 24 h in the presence of DMSO or sudemycin D6. Western blots were performed on cell lysates for Tat and Rev. Data indicate means, and error bars indicate ± SEM ( n = 3). **, P
Figure Legend Snippet: Inhibition of SF3B1 reduces HIV products. (A) U87/CD4/CXCR4 cells were infected with replication-competent HIV-1 for 24 h. Total cellular RNA was obtained, and qRT-PCR was performed for the unspliced (US), singly spliced (SS), and multiply spliced (MS) forms of HIV using specific primers. RNA fold change was calculated relative to DMSO treatment. (B) 293T cells were infected with HIV Δ env for 24 h in the presence of DMSO or sudemycin D6. Western blots were performed on cell lysates for HIV Gag products. Three experiments were quantitated (C). (D) 293T cells were infected with HIV Δ env for 24 h in the presence of DMSO or sudemycin D6. Western blots were performed on cell lysates for Tat and Rev. Data indicate means, and error bars indicate ± SEM ( n = 3). **, P

Techniques Used: Inhibition, Infection, Quantitative RT-PCR, Mass Spectrometry, Western Blot

Inhibition of SF3B1 prevents HIV reactivation from latency. (A) JLAT10.6 cells were incubated with the indicated compounds for 24 h, and FACS was performed to detect live GFP-positive cells as a measure of HIV reactivation from latency. (B) JLAT10.6 cells were incubated with the indicated compounds for 24 h. Cells were lysed and resolved on Western blots for the HIV products. A representative blot of 3 independent experiments is shown. (C) qRT-PCR for a similar experiment in panel B. Fold change is relative to DMSO treatment. (D) Inhibition of HIV reactivation in the Greene model of latency. Resting CD4 + T cells were infected with HIV-1 Luc, treated with protease inhibitor darunavir for 2 days. Cells were incubated with the compounds shown for 24 h in the presence of raltegravir. (See Materials and Methods for details.) HIV reactivation was measured by luciferase-based luminescence in the cell lysates normalized to protein concentration. (E) JLAT10.6 cells were treated with 5 μM sudemycin D6 for 18 h. Cells were washed and allowed to recover for 24 h. At 24, 48, or 72 h after drug washout, the cells were treated with LRAs for 24 h. HIV reactivation was measured with FACS to detect live cells expressing GFP. Data indicate means, and error bars indicate ±SEM ( n = 3). **, P
Figure Legend Snippet: Inhibition of SF3B1 prevents HIV reactivation from latency. (A) JLAT10.6 cells were incubated with the indicated compounds for 24 h, and FACS was performed to detect live GFP-positive cells as a measure of HIV reactivation from latency. (B) JLAT10.6 cells were incubated with the indicated compounds for 24 h. Cells were lysed and resolved on Western blots for the HIV products. A representative blot of 3 independent experiments is shown. (C) qRT-PCR for a similar experiment in panel B. Fold change is relative to DMSO treatment. (D) Inhibition of HIV reactivation in the Greene model of latency. Resting CD4 + T cells were infected with HIV-1 Luc, treated with protease inhibitor darunavir for 2 days. Cells were incubated with the compounds shown for 24 h in the presence of raltegravir. (See Materials and Methods for details.) HIV reactivation was measured by luciferase-based luminescence in the cell lysates normalized to protein concentration. (E) JLAT10.6 cells were treated with 5 μM sudemycin D6 for 18 h. Cells were washed and allowed to recover for 24 h. At 24, 48, or 72 h after drug washout, the cells were treated with LRAs for 24 h. HIV reactivation was measured with FACS to detect live cells expressing GFP. Data indicate means, and error bars indicate ±SEM ( n = 3). **, P

Techniques Used: Inhibition, Incubation, FACS, Western Blot, Quantitative RT-PCR, Infection, Protease Inhibitor, Luciferase, Protein Concentration, Expressing

3) Product Images from "Genetic Confirmation of Mungbean (Vigna radiata) and Mashbean (Vigna mungo) Interspecific Recombinants using Molecular Markers"

Article Title: Genetic Confirmation of Mungbean (Vigna radiata) and Mashbean (Vigna mungo) Interspecific Recombinants using Molecular Markers

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2015.01107

PCR profiles of parental genotypes using SSR markers VR040 (L1–L6), VR062 (L7–L12) and VR0111 (L13–L18). ∗ Marker for recombination .
Figure Legend Snippet: PCR profiles of parental genotypes using SSR markers VR040 (L1–L6), VR062 (L7–L12) and VR0111 (L13–L18). ∗ Marker for recombination .

Techniques Used: Polymerase Chain Reaction, Marker

PCR profiles of parental genotypes along with interspecific recombinants using SSR marker VR040. ∗ Marker for recombination .
Figure Legend Snippet: PCR profiles of parental genotypes along with interspecific recombinants using SSR marker VR040. ∗ Marker for recombination .

Techniques Used: Polymerase Chain Reaction, Marker

PCR profiles of parental genotypes along with interspecific recombinants using SSR marker VR0304. ∗ Marker for recombination .
Figure Legend Snippet: PCR profiles of parental genotypes along with interspecific recombinants using SSR marker VR0304. ∗ Marker for recombination .

Techniques Used: Polymerase Chain Reaction, Marker

PCR profiles of parental genotypes along with interspecific recombinants using SSR marker VR062. ∗ Marker for recombination .
Figure Legend Snippet: PCR profiles of parental genotypes along with interspecific recombinants using SSR marker VR062. ∗ Marker for recombination .

Techniques Used: Polymerase Chain Reaction, Marker

PCR profiles of parental genotypes using RIS-F (A) and RIS-R (B) marker .
Figure Legend Snippet: PCR profiles of parental genotypes using RIS-F (A) and RIS-R (B) marker .

Techniques Used: Polymerase Chain Reaction, Marker

PCR profiles of parental genotypes along with interspecific recombinants using SSR marker VR0111. ∗ Marker for recombination .
Figure Legend Snippet: PCR profiles of parental genotypes along with interspecific recombinants using SSR marker VR0111. ∗ Marker for recombination .

Techniques Used: Polymerase Chain Reaction, Marker

PCR profiles of parental genotypes along with interspecific recombinants using RIS-F. ∗ Marker for recombination .
Figure Legend Snippet: PCR profiles of parental genotypes along with interspecific recombinants using RIS-F. ∗ Marker for recombination .

Techniques Used: Polymerase Chain Reaction, Marker

4) Product Images from "Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential"

Article Title: Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

Journal: Journal of Virology

doi: 10.1128/JVI.01639-17

Induction of LMO2 mRNA expression by FV is increased when the insulator is removed, and induction of LMO2 mRNA expression by LV is decreased when the insulator is added to the LV LTR. cDNA was generated from LMO2 -modified clones containing FV, LV, FV with no insulator (ins.), and LV with the FV insulator placed in the LTR. LMO2 mRNA expression was determined using qRT-PCR. The Hs001534473_m1 primer-probe set and PPIA endogenous control were used to acquire the data. n = 5, 6, 17, and 9 clones, respectively. The error bars indicate SEM.
Figure Legend Snippet: Induction of LMO2 mRNA expression by FV is increased when the insulator is removed, and induction of LMO2 mRNA expression by LV is decreased when the insulator is added to the LV LTR. cDNA was generated from LMO2 -modified clones containing FV, LV, FV with no insulator (ins.), and LV with the FV insulator placed in the LTR. LMO2 mRNA expression was determined using qRT-PCR. The Hs001534473_m1 primer-probe set and PPIA endogenous control were used to acquire the data. n = 5, 6, 17, and 9 clones, respectively. The error bars indicate SEM.

Techniques Used: Expressing, Generated, Modification, Clone Assay, Quantitative RT-PCR

5) Product Images from "Are Bacterial Volatile Compounds Poisonous Odors to a Fungal Pathogen Botrytis cinerea, Alarm Signals to Arabidopsis Seedlings for Eliciting Induced Resistance, or Both?"

Article Title: Are Bacterial Volatile Compounds Poisonous Odors to a Fungal Pathogen Botrytis cinerea, Alarm Signals to Arabidopsis Seedlings for Eliciting Induced Resistance, or Both?

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.00196

Bacillus subtilis GB03 volatile compounds elicit Arabidopsis defense priming of the jasmonic acid and salicylic acid signaling pathways. Defense priming gene expression levels were measured by time-course qRT-PCR analysis of (A) PR1 in the salicylic acid signaling pathway, (B) PDF1.2 in the jasmonic acid signaling pathway, and (C) ChiB in the ethylene signaling pathway (C) . The ratio of gene expression in the B. subtilis GB03-treated plants versus that in the water-treated control relative to expression of the Actin gene is computed as the mean ± SEM. Different letters indicate significant differences between treatments (A,B) according to Fisher’s LSD test at P = 0.05.
Figure Legend Snippet: Bacillus subtilis GB03 volatile compounds elicit Arabidopsis defense priming of the jasmonic acid and salicylic acid signaling pathways. Defense priming gene expression levels were measured by time-course qRT-PCR analysis of (A) PR1 in the salicylic acid signaling pathway, (B) PDF1.2 in the jasmonic acid signaling pathway, and (C) ChiB in the ethylene signaling pathway (C) . The ratio of gene expression in the B. subtilis GB03-treated plants versus that in the water-treated control relative to expression of the Actin gene is computed as the mean ± SEM. Different letters indicate significant differences between treatments (A,B) according to Fisher’s LSD test at P = 0.05.

Techniques Used: Expressing, Quantitative RT-PCR

6) Product Images from "Evaluation of the Importance of VlsE Antigenic Variation for the Enzootic Cycle of Borrelia burgdorferi"

Article Title: Evaluation of the Importance of VlsE Antigenic Variation for the Enzootic Cycle of Borrelia burgdorferi

Journal: PLoS ONE

doi: 10.1371/journal.pone.0124268

Total spirochete loads of vls mutant B . burgdorferi -infected Ixodes scapularis . Spirochetes in unfed nymphs were quantified by qPCR using a primer and internal probe for flaB . The number of spirochetes (log10) for each individual unfed tick is shown as an open circle. The black horizontal bar given for each B . burgdorferi group represents the overall mean. Only PCR-positive samples are included in this Fig. No statistical difference was observed between wtB31 (n = 15) and Δ vlsE (n = 12) or wtB31 (n = 15) and s vlsE (n = 13) in unfed nymphs, as determined by a two tailed t-test.
Figure Legend Snippet: Total spirochete loads of vls mutant B . burgdorferi -infected Ixodes scapularis . Spirochetes in unfed nymphs were quantified by qPCR using a primer and internal probe for flaB . The number of spirochetes (log10) for each individual unfed tick is shown as an open circle. The black horizontal bar given for each B . burgdorferi group represents the overall mean. Only PCR-positive samples are included in this Fig. No statistical difference was observed between wtB31 (n = 15) and Δ vlsE (n = 12) or wtB31 (n = 15) and s vlsE (n = 13) in unfed nymphs, as determined by a two tailed t-test.

Techniques Used: Mutagenesis, Infection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Two Tailed Test

7) Product Images from "Involvement of the adaptor protein 3 complex in lignocellulase secretion in Neurospora crassa revealed by comparative genomic screening"

Article Title: Involvement of the adaptor protein 3 complex in lignocellulase secretion in Neurospora crassa revealed by comparative genomic screening

Journal: Biotechnology for Biofuels

doi: 10.1186/s13068-015-0302-3

Maintenance of high lignocellulase gene expression levels in Δ Ncap3m relative to the wild-type strain (WT) at the late fermentation stage. After Δ Ncap3m and WT conidia were grown on Avicel for 4, 48, 96, or 168 h, the transcriptional abundance of three major lignocellulase genes was evaluated by quantitative real-time PCR (qPCR). The data are normalized to the expression of the WT strain at 48 h for each tested gene, with actin (NCU04173) gene expression levels used as an endogenous control in all samples (standard error of the mean, n = 3). Asterisks indicate significant differences from the control (* P
Figure Legend Snippet: Maintenance of high lignocellulase gene expression levels in Δ Ncap3m relative to the wild-type strain (WT) at the late fermentation stage. After Δ Ncap3m and WT conidia were grown on Avicel for 4, 48, 96, or 168 h, the transcriptional abundance of three major lignocellulase genes was evaluated by quantitative real-time PCR (qPCR). The data are normalized to the expression of the WT strain at 48 h for each tested gene, with actin (NCU04173) gene expression levels used as an endogenous control in all samples (standard error of the mean, n = 3). Asterisks indicate significant differences from the control (* P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

8) Product Images from "Overexpression of PvGF14c from Phyllostachys violascens Delays Flowering Time in Transgenic Arabidopsis"

Article Title: Overexpression of PvGF14c from Phyllostachys violascens Delays Flowering Time in Transgenic Arabidopsis

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.00105

Phenotype analysis of 35S::PvGF14b Arabidopsis plants under LD conditions. (A) Phenotype differences in flowering between 35S::PvGF14b transgenic plants and wild-type plants. Photographs were taken at 26-day-old wild-type plants. (B) The number of rosette leaves in T2 35S::PvGF14b in transgenic plants ( n = 20) and wild-type plants. (C) Bolting time in T2 35S::PvGF14b transgenic plants ( n = 20) and wild-type plants. (D) qRT-PCR expression analysis of PvGF14b . Asterisks “ ∗∗ ” indicate significant differences in comparison with the WT at P
Figure Legend Snippet: Phenotype analysis of 35S::PvGF14b Arabidopsis plants under LD conditions. (A) Phenotype differences in flowering between 35S::PvGF14b transgenic plants and wild-type plants. Photographs were taken at 26-day-old wild-type plants. (B) The number of rosette leaves in T2 35S::PvGF14b in transgenic plants ( n = 20) and wild-type plants. (C) Bolting time in T2 35S::PvGF14b transgenic plants ( n = 20) and wild-type plants. (D) qRT-PCR expression analysis of PvGF14b . Asterisks “ ∗∗ ” indicate significant differences in comparison with the WT at P

Techniques Used: Transgenic Assay, Quantitative RT-PCR, Expressing

Phenotype analysis of 35S::PvGF14e Arabidopsis plants under LD conditions. (A) Phenotype differences in flowering between 35S::PvGF14e transgenic plants and wild-type plants. Photographs were taken at 25-day-old wild-type plants. (B) The number of rosette leaves in T2 35S::PvGF14e in transgenic plants ( n = 20) and wild-type plants. (C) Bolting time in T2 35S::PvGF14e transgenic plants ( n = 20) and wild-type plants. (D) qRT-PCR expression analysis of PvGF14e . Asterisks “ ∗∗ ” indicate significant differences in comparison with the WT at P
Figure Legend Snippet: Phenotype analysis of 35S::PvGF14e Arabidopsis plants under LD conditions. (A) Phenotype differences in flowering between 35S::PvGF14e transgenic plants and wild-type plants. Photographs were taken at 25-day-old wild-type plants. (B) The number of rosette leaves in T2 35S::PvGF14e in transgenic plants ( n = 20) and wild-type plants. (C) Bolting time in T2 35S::PvGF14e transgenic plants ( n = 20) and wild-type plants. (D) qRT-PCR expression analysis of PvGF14e . Asterisks “ ∗∗ ” indicate significant differences in comparison with the WT at P

Techniques Used: Transgenic Assay, Quantitative RT-PCR, Expressing

Phenotype analysis of 35S::PvGF14c Arabidopsis plants under LD conditions. (A) Phenotype differences in flowering between 35S::PvGF14c transgenic plants and wild-type plants. Photographs were taken at 28-day-old wild-type plants. (B) The number of rosette leaves in T3 35S::PvGF14c in transgenic plants ( n = 24) and wild-type plants. (C) Bolting time in T3 35S::PvGF14c transgenic plants ( n = 24) and wild-type plants. (D) qRT-PCR expression analysis of PvGF14c . Asterisks “ ∗∗ ” indicate significant differences in comparison with the WT at P
Figure Legend Snippet: Phenotype analysis of 35S::PvGF14c Arabidopsis plants under LD conditions. (A) Phenotype differences in flowering between 35S::PvGF14c transgenic plants and wild-type plants. Photographs were taken at 28-day-old wild-type plants. (B) The number of rosette leaves in T3 35S::PvGF14c in transgenic plants ( n = 24) and wild-type plants. (C) Bolting time in T3 35S::PvGF14c transgenic plants ( n = 24) and wild-type plants. (D) qRT-PCR expression analysis of PvGF14c . Asterisks “ ∗∗ ” indicate significant differences in comparison with the WT at P

Techniques Used: Transgenic Assay, Quantitative RT-PCR, Expressing

Phenotype analysis of 35S::PvGF14f Arabidopsis plants under LD conditions. (A) Phenotype differences in flowering between 35S::PvGF14f transgenic plants and wild-type plants. Photographs were taken at 27-day-old wild-type plants. (B) The number of rosette leaves in T2 35S::PvGF14f in transgenic plants ( n = 20) and wild-type plants. (C) Bolting time in T2 35S::PvGF14f transgenic plants ( n = 20) and wild-type plants. (D) qRT-PCR expression analysis of PvGF14f .
Figure Legend Snippet: Phenotype analysis of 35S::PvGF14f Arabidopsis plants under LD conditions. (A) Phenotype differences in flowering between 35S::PvGF14f transgenic plants and wild-type plants. Photographs were taken at 27-day-old wild-type plants. (B) The number of rosette leaves in T2 35S::PvGF14f in transgenic plants ( n = 20) and wild-type plants. (C) Bolting time in T2 35S::PvGF14f transgenic plants ( n = 20) and wild-type plants. (D) qRT-PCR expression analysis of PvGF14f .

Techniques Used: Transgenic Assay, Quantitative RT-PCR, Expressing

9) Product Images from "Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody"

Article Title: Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody

Journal: Journal of Cancer

doi: 10.7150/jca.10879

Activation of mitochondrial apoptosis signaling in EMMPRIN down-regulated Caco-2 cells. (A) Detection of Bcl-2, cytochrome c, and caspase-3 mRNA expression levels. Transduced Caco-2 cells were extracted for total RNA and reverse transcribed into cDNA. The expression of Bcl-2 , cytochrome c, and caspase-3 mRNAs were analyzed using real-time RT-PCR analysis. GAPDH mRNA was used as the internal control. The normalized mRNA expression was analyzed using the comparative C T method. The white and black bars represent Ad5/F35- scFv-irrelevant and Ad5/F35- scFv-M6-1B9 transduced Caco-2 cells, respectively. The data are presented as mean±s.e.m. * p
Figure Legend Snippet: Activation of mitochondrial apoptosis signaling in EMMPRIN down-regulated Caco-2 cells. (A) Detection of Bcl-2, cytochrome c, and caspase-3 mRNA expression levels. Transduced Caco-2 cells were extracted for total RNA and reverse transcribed into cDNA. The expression of Bcl-2 , cytochrome c, and caspase-3 mRNAs were analyzed using real-time RT-PCR analysis. GAPDH mRNA was used as the internal control. The normalized mRNA expression was analyzed using the comparative C T method. The white and black bars represent Ad5/F35- scFv-irrelevant and Ad5/F35- scFv-M6-1B9 transduced Caco-2 cells, respectively. The data are presented as mean±s.e.m. * p

Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR

10) Product Images from "Increasing Polyamine Contents Enhances the Stress Tolerance via Reinforcement of Antioxidative Properties"

Article Title: Increasing Polyamine Contents Enhances the Stress Tolerance via Reinforcement of Antioxidative Properties

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2019.01331

S-Adenosylmethionine decarboxylase (SAMDC) transcription and polyamine (PA) contents in transgenic plants with overexpression of SAMDC16. (A) The ratio of each PA in the transgenic plants was determined against the amount of PA contained in the wild-type (WT) plants. (B , C) The amounts of SAMDC16 (B) and PAO (C) were determined by real-time quantitative PCR (qPCR) in WT and transgenic plants in response to salt stress. (D) PA contents, putrescine (Put), spermidine (Spd), spermine (Spm), and total PA were measured by thin-layer chromatography (TLC) after treatment with salt stress. Data are expressed as means ± SD. All data were generated from one representative experiment with three independent biological replicates after verifying the reproducibility of the results in three experiments. An asterisk indicates a significant difference between WT and transgenic plants (* P
Figure Legend Snippet: S-Adenosylmethionine decarboxylase (SAMDC) transcription and polyamine (PA) contents in transgenic plants with overexpression of SAMDC16. (A) The ratio of each PA in the transgenic plants was determined against the amount of PA contained in the wild-type (WT) plants. (B , C) The amounts of SAMDC16 (B) and PAO (C) were determined by real-time quantitative PCR (qPCR) in WT and transgenic plants in response to salt stress. (D) PA contents, putrescine (Put), spermidine (Spd), spermine (Spm), and total PA were measured by thin-layer chromatography (TLC) after treatment with salt stress. Data are expressed as means ± SD. All data were generated from one representative experiment with three independent biological replicates after verifying the reproducibility of the results in three experiments. An asterisk indicates a significant difference between WT and transgenic plants (* P

Techniques Used: Transgenic Assay, Over Expression, Real-time Polymerase Chain Reaction, Thin Layer Chromatography, Generated

11) Product Images from "Aldo-Keto Reductases in the Eye"

Article Title: Aldo-Keto Reductases in the Eye

Journal: Journal of Ophthalmology

doi: 10.1155/2010/521204

Expression of AKR1B1 and AKR1B10 in human eye tissues. Gene transcript levels were measured by quantitative real-time PCR as described in Section 2 . Data are mean ± SD among 5 nondiabetic male donors aged 65. ± 9.2 years. Data for AKR gene transcripts levels are normalized to RT-PCR for β -actin.
Figure Legend Snippet: Expression of AKR1B1 and AKR1B10 in human eye tissues. Gene transcript levels were measured by quantitative real-time PCR as described in Section 2 . Data are mean ± SD among 5 nondiabetic male donors aged 65. ± 9.2 years. Data for AKR gene transcripts levels are normalized to RT-PCR for β -actin.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

12) Product Images from "Osteoprotegerin mediates tumor-promoting effects of Interleukin-1beta in breast cancer cells"

Article Title: Osteoprotegerin mediates tumor-promoting effects of Interleukin-1beta in breast cancer cells

Journal: Molecular Cancer

doi: 10.1186/s12943-017-0606-y

Co-culture with THP-1 macrophages induces OPG secretion in breast cancer cells. a Diagram of the transwell co-culture experiment set-up. Breast cancer cells were co-cultured with THP-1 macrophages in the presence and absence of Interleukin-1 receptor antagonist (IL-1RA, 400 ng/mL). b After 8 h of co-culture, relative OPG mRNA was measured by qRT-PCR, ( n ≥ 3). c Breast cancer cells were subsequently cultured alone in fresh media for an additional 16 h and OPG secreted protein was measured from this supernatant by ELISA, ( n ≥ 3). OPG mRNA and secreted protein levels reveal that the co-culture mediated induction of OPG expression in breast cancer cells were partially repressed by IL-1RA. d Representative image for d CD68 immunohistochemistry and e OPG immunohistochemistry on primary/malignant tumor sections on a human breast cancer tissue microarray. Images are taken at 400× magnification. Data are represented by mean ± SD. Asterisks indicate statistical significance ( p
Figure Legend Snippet: Co-culture with THP-1 macrophages induces OPG secretion in breast cancer cells. a Diagram of the transwell co-culture experiment set-up. Breast cancer cells were co-cultured with THP-1 macrophages in the presence and absence of Interleukin-1 receptor antagonist (IL-1RA, 400 ng/mL). b After 8 h of co-culture, relative OPG mRNA was measured by qRT-PCR, ( n ≥ 3). c Breast cancer cells were subsequently cultured alone in fresh media for an additional 16 h and OPG secreted protein was measured from this supernatant by ELISA, ( n ≥ 3). OPG mRNA and secreted protein levels reveal that the co-culture mediated induction of OPG expression in breast cancer cells were partially repressed by IL-1RA. d Representative image for d CD68 immunohistochemistry and e OPG immunohistochemistry on primary/malignant tumor sections on a human breast cancer tissue microarray. Images are taken at 400× magnification. Data are represented by mean ± SD. Asterisks indicate statistical significance ( p

Techniques Used: Co-Culture Assay, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry, Microarray

IL1B promotes the invasion of breast cancer cells in an OPG-dependent manner. MDA-MB-436 breast cancer cells transfected with OPG (siOPG1) or negative control (siNeg) siRNA were pretreated with IL1B (10 ng/mL), and subsequently assayed for invasiveness. a IL1B-mediated cell invasion is reduced in OPG knockdown cells, ( n = 3). Data is represented as relative fluorescence units (RFU) fold change relative to the untreated negative control. b OPG knockdown in the breast cancer cells used in the cell invasion assay was assessed at the end point of the experiment. Knockdown was verified by qRT-PCR. MDA-MB-436 cells transfected with OPG or control siRNA were treated with IL1B or PBS (10 ng/mL) for 72 h. c OPG knockdown at 72 h was verified by qRT-PCR. Assessment of d MMP3 and e IL1B mRNA levels indicate the IL1B-mediated induction is inhibited by OPG depletion, ( n = 3). Data are represented by mean ± SD. Asterisks indicate statistical significance ( p
Figure Legend Snippet: IL1B promotes the invasion of breast cancer cells in an OPG-dependent manner. MDA-MB-436 breast cancer cells transfected with OPG (siOPG1) or negative control (siNeg) siRNA were pretreated with IL1B (10 ng/mL), and subsequently assayed for invasiveness. a IL1B-mediated cell invasion is reduced in OPG knockdown cells, ( n = 3). Data is represented as relative fluorescence units (RFU) fold change relative to the untreated negative control. b OPG knockdown in the breast cancer cells used in the cell invasion assay was assessed at the end point of the experiment. Knockdown was verified by qRT-PCR. MDA-MB-436 cells transfected with OPG or control siRNA were treated with IL1B or PBS (10 ng/mL) for 72 h. c OPG knockdown at 72 h was verified by qRT-PCR. Assessment of d MMP3 and e IL1B mRNA levels indicate the IL1B-mediated induction is inhibited by OPG depletion, ( n = 3). Data are represented by mean ± SD. Asterisks indicate statistical significance ( p

Techniques Used: Multiple Displacement Amplification, Transfection, Negative Control, Fluorescence, Invasion Assay, Quantitative RT-PCR

IL1B induces OPG secretion in breast cancer cell lines regardless of subtype and basal OPG protein levels. a Basal OPG and b IL1B protein levels were assessed by ELISA on supernatant collected from several different breast cancer cell lines, ( n ≥ 3). c Relative OPG mRNA measured by qRT-PCR and d OPG secreted protein levels increase upon treatment with IL1B (10 ng/mL) in several different breast cancer cell lines, ( n ≥ 3). e OPG RNA and f OPG secreted protein levels decrease upon treatment with PBS or IL-1B receptor antagonist IL-1RA (50 ng/mL) of MDA-MB-436 cells ( n = 4). Data are represented by mean ± SD. Asterisks indicate statistical significance ( p
Figure Legend Snippet: IL1B induces OPG secretion in breast cancer cell lines regardless of subtype and basal OPG protein levels. a Basal OPG and b IL1B protein levels were assessed by ELISA on supernatant collected from several different breast cancer cell lines, ( n ≥ 3). c Relative OPG mRNA measured by qRT-PCR and d OPG secreted protein levels increase upon treatment with IL1B (10 ng/mL) in several different breast cancer cell lines, ( n ≥ 3). e OPG RNA and f OPG secreted protein levels decrease upon treatment with PBS or IL-1B receptor antagonist IL-1RA (50 ng/mL) of MDA-MB-436 cells ( n = 4). Data are represented by mean ± SD. Asterisks indicate statistical significance ( p

Techniques Used: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Multiple Displacement Amplification

13) Product Images from "Temperature Oscillation Modulated Self-Assembly of Periodic Concentric Layered Magnesium Carbonate Microparticles"

Article Title: Temperature Oscillation Modulated Self-Assembly of Periodic Concentric Layered Magnesium Carbonate Microparticles

Journal: PLoS ONE

doi: 10.1371/journal.pone.0088648

Magnesium carbonate microhemispheres synthesized with a programmable PCR thermocycler. The reactant was a mixture of 240 2 and 500 mM NaHCO 3 (1∶1, V/V). (A) Similar temperature oscillation patterns having different periods. (B–D) Transmitted light microscopic images of microhemispheres produced by the temperature oscillation patterns with 60, 30 and 15-min period, respectively. The corresponding layer thicknesses were 12.0±0.2, 6.0±0.1 and 2.6±0.0 µm.
Figure Legend Snippet: Magnesium carbonate microhemispheres synthesized with a programmable PCR thermocycler. The reactant was a mixture of 240 2 and 500 mM NaHCO 3 (1∶1, V/V). (A) Similar temperature oscillation patterns having different periods. (B–D) Transmitted light microscopic images of microhemispheres produced by the temperature oscillation patterns with 60, 30 and 15-min period, respectively. The corresponding layer thicknesses were 12.0±0.2, 6.0±0.1 and 2.6±0.0 µm.

Techniques Used: Synthesized, Polymerase Chain Reaction, Produced

14) Product Images from "Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data"

Article Title: Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data

Journal: Scientific Reports

doi: 10.1038/s41598-017-02217-x

Assessment of primer efficiency, linear dynamic range and precision of ddPCR and qPCR platforms for low target concentration. Five reactions of 45 µL were prepared in triplicate from 1/2 serial dilutions of synthetic DNA in nuclease-free water with primers and ddPCR EvaGreen supermix. Each reaction mix was split for quantification in qPCR and ddPCR (20 µL for each platform). Amplification traces ( A ), standard curve (A inset), melt analysis ( B ) and tabulated relative fold difference (ΔCq) results ( C ) for qPCR. The ddPCR amplitude plot ( D ) and tabulated absolute concentration data ( E ). NTC: No Template Control; Dilution: Dilution factor of DNA samples; Rep: Replicate number; Avg: Average of the replicates; Std. Dev.: Standard Deviation between the replicates; % CV: % Coefficient of Variance (Std. Dev./Avg*100).
Figure Legend Snippet: Assessment of primer efficiency, linear dynamic range and precision of ddPCR and qPCR platforms for low target concentration. Five reactions of 45 µL were prepared in triplicate from 1/2 serial dilutions of synthetic DNA in nuclease-free water with primers and ddPCR EvaGreen supermix. Each reaction mix was split for quantification in qPCR and ddPCR (20 µL for each platform). Amplification traces ( A ), standard curve (A inset), melt analysis ( B ) and tabulated relative fold difference (ΔCq) results ( C ) for qPCR. The ddPCR amplitude plot ( D ) and tabulated absolute concentration data ( E ). NTC: No Template Control; Dilution: Dilution factor of DNA samples; Rep: Replicate number; Avg: Average of the replicates; Std. Dev.: Standard Deviation between the replicates; % CV: % Coefficient of Variance (Std. Dev./Avg*100).

Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Amplification, Standard Deviation

Effect of consistent sample contamination between identical DNA dilutions for ddPCR and qPCR technologies. Four reactions of 50 µL were prepared in triplicate from 1/2 serial dilutions of synthetic DNA in a background of either 10 µL ( A ) or 12.5 µL ( B ) of 1X reverse transcription (RT) mix with primers and ddPCR EvaGreen supermix. Each reaction mix was split for quantification in qPCR and ddPCR (20 µL for each platform containing either 4 µL or 5 µL of contaminating RT mix) and run on a single plate for each platform. Amplification traces ( A and C ), standard curves ( B and D ) and tabulated relative fold difference (ΔCq) results ( E and F ) were generated for qPCR. The ddPCR amplitude plots ( G and I ) and tabulated absolute concentration data ( H and J ) from the same reactions supplemented with 4 uL ( A,B,E,G,H ) and 5 uL ( C,D,F,I,J ) of RT mix were produced. NTC: No Template Control; Dilution: Dilution factor of DNA samples; Rep: Replicate number; Avg: Average of the replicates; Std. Dev.: Standard Deviation between the replicates; % CV: % Coefficient of Variance (Std. Dev./Avg*100).
Figure Legend Snippet: Effect of consistent sample contamination between identical DNA dilutions for ddPCR and qPCR technologies. Four reactions of 50 µL were prepared in triplicate from 1/2 serial dilutions of synthetic DNA in a background of either 10 µL ( A ) or 12.5 µL ( B ) of 1X reverse transcription (RT) mix with primers and ddPCR EvaGreen supermix. Each reaction mix was split for quantification in qPCR and ddPCR (20 µL for each platform containing either 4 µL or 5 µL of contaminating RT mix) and run on a single plate for each platform. Amplification traces ( A and C ), standard curves ( B and D ) and tabulated relative fold difference (ΔCq) results ( E and F ) were generated for qPCR. The ddPCR amplitude plots ( G and I ) and tabulated absolute concentration data ( H and J ) from the same reactions supplemented with 4 uL ( A,B,E,G,H ) and 5 uL ( C,D,F,I,J ) of RT mix were produced. NTC: No Template Control; Dilution: Dilution factor of DNA samples; Rep: Replicate number; Avg: Average of the replicates; Std. Dev.: Standard Deviation between the replicates; % CV: % Coefficient of Variance (Std. Dev./Avg*100).

Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Generated, Concentration Assay, Produced, Standard Deviation

15) Product Images from "Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements"

Article Title: Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements

Journal: Mutagenesis

doi: 10.1093/mutage/geq048

Summary of RT–PCR analysis of in vivo transgene copy number. Standard curves were based on quantifications of the ori amplicons, using replicated sets of template composed of CD2F 1 non-transgenic mouse DNA mixed with λgt10- lacZ (see Figure 3 Materials and methods) to generate haploid standards of 1, 2.5, 5, 10, 20, 40 and 50 copies. Cycle thresholds ( C t ) and efficiency parameters were obtained by iCycler software and show a linear correlation with log copy number values ( R 2 = 0.9924). Shown are average C t values for: ori standards (open circles), single-copy annexin V exon 4 (closed triangle, A); R5 rearrangement (closed circle, R5); interpolated copies of Muta™mouse ori (closed diamond, MM) and 18S (closed box, 18S).
Figure Legend Snippet: Summary of RT–PCR analysis of in vivo transgene copy number. Standard curves were based on quantifications of the ori amplicons, using replicated sets of template composed of CD2F 1 non-transgenic mouse DNA mixed with λgt10- lacZ (see Figure 3 Materials and methods) to generate haploid standards of 1, 2.5, 5, 10, 20, 40 and 50 copies. Cycle thresholds ( C t ) and efficiency parameters were obtained by iCycler software and show a linear correlation with log copy number values ( R 2 = 0.9924). Shown are average C t values for: ori standards (open circles), single-copy annexin V exon 4 (closed triangle, A); R5 rearrangement (closed circle, R5); interpolated copies of Muta™mouse ori (closed diamond, MM) and 18S (closed box, 18S).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vivo, Transgenic Assay, Software

Model of the Muta™Mouse λgt10- lacZ transgene derived by sequence analysis. The transgene monomer has 47 513 bp and 57 ORFs and is based on nucleotide sequencing of PCR amplicons derived from systematic scanning of functional regions of λgt10- lacZ using Muta™Mouse genomic DNA from tissues of both gender and λgt10- lacZ in vivo copies rescued by in vitro phage packaging and commercial stocks of λCI857 and λgt10. Data include the b527 deletion and unfinished parts of bacteriophage imm434 (accession numbers M60848, Y00118) and the 5.2 kb EcoR1-DraI fragment of pMC1511, which contains the E.coli lacZ mutation reporter (GenBank® L08935) (hatched-box). The GenBank® accession numbers for sequences other than those reported already for λ (NC_001416; 48.5 kb) are given in Table II . Functional regions included left (COS L ) and right (COS R ) cohesive ends and ORFs required for virion assembly and DNA packaging and origin of λ DNA replication (ORI). Arrows show orientation of ORFs common to the 48.5 kb λ bacteriophage as identified by λ gene nomenclature (see NC_001416). Novel in vivo copy rearrangements are described in Figure 2 and Table II . The large open box spans a novel finding of a region of substitution by lambdoid Rac prophage tail fiber assembly gene ( lom and ORFs 401, 314 and 194) found also in λgt10 and λCI857 commercial stocks. Also identified are crossover hot spot instigator motifs (Chi sites) conveying potential for lacZ recombination with the E.coli genome. The symbol χ + signifies a fully functional Chi motif (starts at nucleotide position 3508 on the reverse or antistrand with respect to E.coli lacZ (accession J01636). χ 0 signifies a one base difference version of the Chi motif that requires a single mutation for full function. These χ 0 sites start at nucleotide positions 3149 and 3152 for the two on the anti-strand and position 3248 for the one on the sense strand. MAR-1 and MAR-2 show location of sequences identified as matrix-associated regions using a computational approach ( 15 ). MAR-1 is located 5′ to the lambda INT gene and MAR-2 is located in the 3′ non-coding λgt-10 region and they represent regions that may contribute to higher order mammalian chromosome loop structures. In a head-to-tail arrangement of transgenes, MAR-2 would be located within 1.4 kb of the COS L site and in the vicinity of rearrangements featured in Figure 2 .
Figure Legend Snippet: Model of the Muta™Mouse λgt10- lacZ transgene derived by sequence analysis. The transgene monomer has 47 513 bp and 57 ORFs and is based on nucleotide sequencing of PCR amplicons derived from systematic scanning of functional regions of λgt10- lacZ using Muta™Mouse genomic DNA from tissues of both gender and λgt10- lacZ in vivo copies rescued by in vitro phage packaging and commercial stocks of λCI857 and λgt10. Data include the b527 deletion and unfinished parts of bacteriophage imm434 (accession numbers M60848, Y00118) and the 5.2 kb EcoR1-DraI fragment of pMC1511, which contains the E.coli lacZ mutation reporter (GenBank® L08935) (hatched-box). The GenBank® accession numbers for sequences other than those reported already for λ (NC_001416; 48.5 kb) are given in Table II . Functional regions included left (COS L ) and right (COS R ) cohesive ends and ORFs required for virion assembly and DNA packaging and origin of λ DNA replication (ORI). Arrows show orientation of ORFs common to the 48.5 kb λ bacteriophage as identified by λ gene nomenclature (see NC_001416). Novel in vivo copy rearrangements are described in Figure 2 and Table II . The large open box spans a novel finding of a region of substitution by lambdoid Rac prophage tail fiber assembly gene ( lom and ORFs 401, 314 and 194) found also in λgt10 and λCI857 commercial stocks. Also identified are crossover hot spot instigator motifs (Chi sites) conveying potential for lacZ recombination with the E.coli genome. The symbol χ + signifies a fully functional Chi motif (starts at nucleotide position 3508 on the reverse or antistrand with respect to E.coli lacZ (accession J01636). χ 0 signifies a one base difference version of the Chi motif that requires a single mutation for full function. These χ 0 sites start at nucleotide positions 3149 and 3152 for the two on the anti-strand and position 3248 for the one on the sense strand. MAR-1 and MAR-2 show location of sequences identified as matrix-associated regions using a computational approach ( 15 ). MAR-1 is located 5′ to the lambda INT gene and MAR-2 is located in the 3′ non-coding λgt-10 region and they represent regions that may contribute to higher order mammalian chromosome loop structures. In a head-to-tail arrangement of transgenes, MAR-2 would be located within 1.4 kb of the COS L site and in the vicinity of rearrangements featured in Figure 2 .

Techniques Used: Derivative Assay, Sequencing, Polymerase Chain Reaction, Functional Assay, In Vivo, In Vitro, Mutagenesis

16) Product Images from "Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements"

Article Title: Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements

Journal: Mutagenesis

doi: 10.1093/mutage/geq048

Summary of RT–PCR analysis of in vivo transgene copy number. Standard curves were based on quantifications of the ori amplicons, using replicated sets of template composed of CD2F 1 non-transgenic mouse DNA mixed with λgt10- lacZ (see Figure 3 Materials and methods) to generate haploid standards of 1, 2.5, 5, 10, 20, 40 and 50 copies. Cycle thresholds ( C t ) and efficiency parameters were obtained by iCycler software and show a linear correlation with log copy number values ( R 2 = 0.9924). Shown are average C t values for: ori standards (open circles), single-copy annexin V exon 4 (closed triangle, A); R5 rearrangement (closed circle, R5); interpolated copies of Muta™mouse ori (closed diamond, MM) and 18S (closed box, 18S).
Figure Legend Snippet: Summary of RT–PCR analysis of in vivo transgene copy number. Standard curves were based on quantifications of the ori amplicons, using replicated sets of template composed of CD2F 1 non-transgenic mouse DNA mixed with λgt10- lacZ (see Figure 3 Materials and methods) to generate haploid standards of 1, 2.5, 5, 10, 20, 40 and 50 copies. Cycle thresholds ( C t ) and efficiency parameters were obtained by iCycler software and show a linear correlation with log copy number values ( R 2 = 0.9924). Shown are average C t values for: ori standards (open circles), single-copy annexin V exon 4 (closed triangle, A); R5 rearrangement (closed circle, R5); interpolated copies of Muta™mouse ori (closed diamond, MM) and 18S (closed box, 18S).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vivo, Transgenic Assay, Software

Model of the Muta™Mouse λgt10- lacZ transgene derived by sequence analysis. The transgene monomer has 47 513 bp and 57 ORFs and is based on nucleotide sequencing of PCR amplicons derived from systematic scanning of functional regions of λgt10- lacZ using Muta™Mouse genomic DNA from tissues of both gender and λgt10- lacZ in vivo copies rescued by in vitro phage packaging and commercial stocks of λCI857 and λgt10. Data include the b527 deletion and unfinished parts of bacteriophage imm434 (accession numbers M60848, Y00118) and the 5.2 kb EcoR1-DraI fragment of pMC1511, which contains the E.coli lacZ mutation reporter (GenBank® L08935) (hatched-box). The GenBank® accession numbers for sequences other than those reported already for λ (NC_001416; 48.5 kb) are given in Table II . Functional regions included left (COS L ) and right (COS R ) cohesive ends and ORFs required for virion assembly and DNA packaging and origin of λ DNA replication (ORI). Arrows show orientation of ORFs common to the 48.5 kb λ bacteriophage as identified by λ gene nomenclature (see NC_001416). Novel in vivo copy rearrangements are described in Figure 2 and Table II . The large open box spans a novel finding of a region of substitution by lambdoid Rac prophage tail fiber assembly gene ( lom and ORFs 401, 314 and 194) found also in λgt10 and λCI857 commercial stocks. Also identified are crossover hot spot instigator motifs (Chi sites) conveying potential for lacZ recombination with the E.coli genome. The symbol χ + signifies a fully functional Chi motif (starts at nucleotide position 3508 on the reverse or antistrand with respect to E.coli lacZ (accession J01636). χ 0 signifies a one base difference version of the Chi motif that requires a single mutation for full function. These χ 0 sites start at nucleotide positions 3149 and 3152 for the two on the anti-strand and position 3248 for the one on the sense strand. MAR-1 and MAR-2 show location of sequences identified as matrix-associated regions using a computational approach ( 15 ). MAR-1 is located 5′ to the lambda INT gene and MAR-2 is located in the 3′ non-coding λgt-10 region and they represent regions that may contribute to higher order mammalian chromosome loop structures. In a head-to-tail arrangement of transgenes, MAR-2 would be located within 1.4 kb of the COS L site and in the vicinity of rearrangements featured in Figure 2 .
Figure Legend Snippet: Model of the Muta™Mouse λgt10- lacZ transgene derived by sequence analysis. The transgene monomer has 47 513 bp and 57 ORFs and is based on nucleotide sequencing of PCR amplicons derived from systematic scanning of functional regions of λgt10- lacZ using Muta™Mouse genomic DNA from tissues of both gender and λgt10- lacZ in vivo copies rescued by in vitro phage packaging and commercial stocks of λCI857 and λgt10. Data include the b527 deletion and unfinished parts of bacteriophage imm434 (accession numbers M60848, Y00118) and the 5.2 kb EcoR1-DraI fragment of pMC1511, which contains the E.coli lacZ mutation reporter (GenBank® L08935) (hatched-box). The GenBank® accession numbers for sequences other than those reported already for λ (NC_001416; 48.5 kb) are given in Table II . Functional regions included left (COS L ) and right (COS R ) cohesive ends and ORFs required for virion assembly and DNA packaging and origin of λ DNA replication (ORI). Arrows show orientation of ORFs common to the 48.5 kb λ bacteriophage as identified by λ gene nomenclature (see NC_001416). Novel in vivo copy rearrangements are described in Figure 2 and Table II . The large open box spans a novel finding of a region of substitution by lambdoid Rac prophage tail fiber assembly gene ( lom and ORFs 401, 314 and 194) found also in λgt10 and λCI857 commercial stocks. Also identified are crossover hot spot instigator motifs (Chi sites) conveying potential for lacZ recombination with the E.coli genome. The symbol χ + signifies a fully functional Chi motif (starts at nucleotide position 3508 on the reverse or antistrand with respect to E.coli lacZ (accession J01636). χ 0 signifies a one base difference version of the Chi motif that requires a single mutation for full function. These χ 0 sites start at nucleotide positions 3149 and 3152 for the two on the anti-strand and position 3248 for the one on the sense strand. MAR-1 and MAR-2 show location of sequences identified as matrix-associated regions using a computational approach ( 15 ). MAR-1 is located 5′ to the lambda INT gene and MAR-2 is located in the 3′ non-coding λgt-10 region and they represent regions that may contribute to higher order mammalian chromosome loop structures. In a head-to-tail arrangement of transgenes, MAR-2 would be located within 1.4 kb of the COS L site and in the vicinity of rearrangements featured in Figure 2 .

Techniques Used: Derivative Assay, Sequencing, Polymerase Chain Reaction, Functional Assay, In Vivo, In Vitro, Mutagenesis

17) Product Images from "Structural and biochemical characterization of the Cutibacterium acnes exo-β-1,4-mannosidase that targets the N-glycan core of host glycoproteins"

Article Title: Structural and biochemical characterization of the Cutibacterium acnes exo-β-1,4-mannosidase that targets the N-glycan core of host glycoproteins

Journal: PLoS ONE

doi: 10.1371/journal.pone.0204703

Close up of the active site in Ca Man5_18. (A) Interactions made by the catalytic residues Glu140 and Glu259 with Arg41, Asn139, His215, Trp217 and Trp295. (B) Hypothetical binding of Man-GlcNAc 2 and possible interactions with Ser88, Asn139 and His151. Purple dashed lines highlight interactions.
Figure Legend Snippet: Close up of the active site in Ca Man5_18. (A) Interactions made by the catalytic residues Glu140 and Glu259 with Arg41, Asn139, His215, Trp217 and Trp295. (B) Hypothetical binding of Man-GlcNAc 2 and possible interactions with Ser88, Asn139 and His151. Purple dashed lines highlight interactions.

Techniques Used: Binding Assay

Ca Man5_18 activity on p NP- β Man. Dependency of Ca Man5_18 activity with p NP- β Man as substrate on (A) temperature, and on (B) pH. (C) Kinetic stability of Ca Man5_18 hydrolysis of p NP- β Man. Symbols: filled circles, 40°C; empty circles, 50°C; filled triangles, 60°C; empty triangles, 70°C. (D) ThermoFluor analysis of thermal stability to unfolding as a function of pH.
Figure Legend Snippet: Ca Man5_18 activity on p NP- β Man. Dependency of Ca Man5_18 activity with p NP- β Man as substrate on (A) temperature, and on (B) pH. (C) Kinetic stability of Ca Man5_18 hydrolysis of p NP- β Man. Symbols: filled circles, 40°C; empty circles, 50°C; filled triangles, 60°C; empty triangles, 70°C. (D) ThermoFluor analysis of thermal stability to unfolding as a function of pH.

Techniques Used: Activity Assay

Ribbon representation of the Ca Man5_18 subunit structure. The subunit structure of Ca Man5_18 features a (β/α) 8 TIM-barrel fold typical for members of the GH5 family. The catalytic acid/base Glu140 and the nucleophile Glu259 are represented as stick models. Secondary-structure elements are represented as: α-helices, blue cylinders; β-sheets, red arrows; and loops, gray coils.
Figure Legend Snippet: Ribbon representation of the Ca Man5_18 subunit structure. The subunit structure of Ca Man5_18 features a (β/α) 8 TIM-barrel fold typical for members of the GH5 family. The catalytic acid/base Glu140 and the nucleophile Glu259 are represented as stick models. Secondary-structure elements are represented as: α-helices, blue cylinders; β-sheets, red arrows; and loops, gray coils.

Techniques Used:

Dimerization of Ca Man5_18. The dimer interface between subunit A (cyan) and subunit B (brown) with the side chains highlighted that stabilize the dimeric state through intersubunit salt links (Asp19-Arg57 and Arg53-Asp306). Loops L2 (red; residues 89–92) and L3 (blue; residues 92–102) participate in dimer formation, as well as in forming the blockage of the active site required for exo-mode activity. Inset: Interactions formed by loops L2 and L3 in the active site. The steric blockage is mainly provided by L2, supported by L3 and L5 (residues 299–314). The catalytic residues Glu140 and Glu259 are colored yellow.
Figure Legend Snippet: Dimerization of Ca Man5_18. The dimer interface between subunit A (cyan) and subunit B (brown) with the side chains highlighted that stabilize the dimeric state through intersubunit salt links (Asp19-Arg57 and Arg53-Asp306). Loops L2 (red; residues 89–92) and L3 (blue; residues 92–102) participate in dimer formation, as well as in forming the blockage of the active site required for exo-mode activity. Inset: Interactions formed by loops L2 and L3 in the active site. The steric blockage is mainly provided by L2, supported by L3 and L5 (residues 299–314). The catalytic residues Glu140 and Glu259 are colored yellow.

Techniques Used: Activity Assay

TLC analysis of product formation. Separation of the hydrolysis products from time-dependent hydrolysis of mannooligosaccharides and Man-GlcNAc by wild-type Ca Man5_18. (A) M3 and M4; (B) M6 and M5 with the mannooligosaccharide standards M1-M6 at the left; (C) Man-GlcNAc hydrolysis by Ca Man5_18 wild type and the E140Q/E259Q double mutant.
Figure Legend Snippet: TLC analysis of product formation. Separation of the hydrolysis products from time-dependent hydrolysis of mannooligosaccharides and Man-GlcNAc by wild-type Ca Man5_18. (A) M3 and M4; (B) M6 and M5 with the mannooligosaccharide standards M1-M6 at the left; (C) Man-GlcNAc hydrolysis by Ca Man5_18 wild type and the E140Q/E259Q double mutant.

Techniques Used: Thin Layer Chromatography, Mutagenesis

18) Product Images from "Characterization of a thaumarchaeal symbiont that drives incomplete nitrification in the tropical sponge Ianthella basta"

Article Title: Characterization of a thaumarchaeal symbiont that drives incomplete nitrification in the tropical sponge Ianthella basta

Journal: bioRxiv

doi: 10.1101/527234

Phylogeny of Ca. Nitrosospongia bastadiensis. (A) Bayesian 16S rRNA gene tree. (B) Bayesian amoA gene tree. (C) Bayesian phylogenomic tree. For this analysis, Ca . N. bastadiensis and Ca. C. symbiosum amoA sequences were placed into a reference tree, representing all OTU representatives of the curated database provided in Alves et al. (2018) , using the Evolutionary Placement Algorithm (EPA; Berger et al ., 2011) implemented in RAxML-HPC 8.2.11 ( Stamatakis, 2014 ). Representative sequences clustering with the sponge symbiont amoA sequences were then analyzed along with amoA sequences from the other genome-sequenced AOA. Note that Ca. Nitrosotalea okcheonensis possesses two amoA gene copies (Herbold et al ., 2017). (C) Bayesian phylogenomic tree based on 34 concatenated universal, single-copy marker genes identified with CheckM ( Parks et al ., 2015 ). Bayesian posterior support values > 0.5 are indicated for each branch. Outgroups for all trees consisted of all three genome-sequenced members of the Nitrososphaera cluster, both members of the Nitrosocosmicus clade, and Ca. Nitrosocaldus icelandicus. In all trees, sequences obtained from sponges are depicted in bold.
Figure Legend Snippet: Phylogeny of Ca. Nitrosospongia bastadiensis. (A) Bayesian 16S rRNA gene tree. (B) Bayesian amoA gene tree. (C) Bayesian phylogenomic tree. For this analysis, Ca . N. bastadiensis and Ca. C. symbiosum amoA sequences were placed into a reference tree, representing all OTU representatives of the curated database provided in Alves et al. (2018) , using the Evolutionary Placement Algorithm (EPA; Berger et al ., 2011) implemented in RAxML-HPC 8.2.11 ( Stamatakis, 2014 ). Representative sequences clustering with the sponge symbiont amoA sequences were then analyzed along with amoA sequences from the other genome-sequenced AOA. Note that Ca. Nitrosotalea okcheonensis possesses two amoA gene copies (Herbold et al ., 2017). (C) Bayesian phylogenomic tree based on 34 concatenated universal, single-copy marker genes identified with CheckM ( Parks et al ., 2015 ). Bayesian posterior support values > 0.5 are indicated for each branch. Outgroups for all trees consisted of all three genome-sequenced members of the Nitrososphaera cluster, both members of the Nitrosocosmicus clade, and Ca. Nitrosocaldus icelandicus. In all trees, sequences obtained from sponges are depicted in bold.

Techniques Used: Marker

19) Product Images from "Prognostic value of circulating tumour DNA in patients undergoing curative resection for pancreatic cancer"

Article Title: Prognostic value of circulating tumour DNA in patients undergoing curative resection for pancreatic cancer

Journal: British Journal of Cancer

doi: 10.1038/bjc.2016.175

Overview of droplet digital PCR assay. ( A ) Schematic representation of the droplet digital PCR (ddPCR) assay, which is based on nanolitre-sized water-in-oil emulsion droplet technology. In this assay, target DNA molecules are uniformly distributed across thousands of emulsified droplets, after which PCR amplification is performed in each partitioned droplet. After amplification, reactions containing one or more target DNA molecules represent the positive end-point, whereas those without target DNA molecules represent the negative end-point. The number of target DNA molecules present can be calculated from the fraction of positive end-point reactions using Poisson statistics. ( B ) Two-dimensional histogram of ddPCR assay for KRAS amplification. FAM (blue) and HEX (green) fluorescence levels were plotted for each droplet. Clusters in the upper and right halves of the plot (dashed circle and solid circle) represent the positive mutant and wild-type KRAS end-point results, respectively.
Figure Legend Snippet: Overview of droplet digital PCR assay. ( A ) Schematic representation of the droplet digital PCR (ddPCR) assay, which is based on nanolitre-sized water-in-oil emulsion droplet technology. In this assay, target DNA molecules are uniformly distributed across thousands of emulsified droplets, after which PCR amplification is performed in each partitioned droplet. After amplification, reactions containing one or more target DNA molecules represent the positive end-point, whereas those without target DNA molecules represent the negative end-point. The number of target DNA molecules present can be calculated from the fraction of positive end-point reactions using Poisson statistics. ( B ) Two-dimensional histogram of ddPCR assay for KRAS amplification. FAM (blue) and HEX (green) fluorescence levels were plotted for each droplet. Clusters in the upper and right halves of the plot (dashed circle and solid circle) represent the positive mutant and wild-type KRAS end-point results, respectively.

Techniques Used: Digital PCR, Polymerase Chain Reaction, Amplification, Fluorescence, Mutagenesis

20) Product Images from "Splicing Factor 3B Subunit 1 Interacts with HIV Tat and Plays a Role in Viral Transcription and Reactivation from Latency"

Article Title: Splicing Factor 3B Subunit 1 Interacts with HIV Tat and Plays a Role in Viral Transcription and Reactivation from Latency

Journal: mBio

doi: 10.1128/mBio.01423-18

Inhibition of SF3B1 reduces HIV products. (A) U87/CD4/CXCR4 cells were infected with replication-competent HIV-1 for 24 h. Total cellular RNA was obtained, and qRT-PCR was performed for the unspliced (US), singly spliced (SS), and multiply spliced (MS) forms of HIV using specific primers. RNA fold change was calculated relative to DMSO treatment. (B) 293T cells were infected with HIV Δ env for 24 h in the presence of DMSO or sudemycin D6. Western blots were performed on cell lysates for HIV Gag products. Three experiments were quantitated (C). (D) 293T cells were infected with HIV Δ env for 24 h in the presence of DMSO or sudemycin D6. Western blots were performed on cell lysates for Tat and Rev. Data indicate means, and error bars indicate ± SEM ( n = 3). **, P
Figure Legend Snippet: Inhibition of SF3B1 reduces HIV products. (A) U87/CD4/CXCR4 cells were infected with replication-competent HIV-1 for 24 h. Total cellular RNA was obtained, and qRT-PCR was performed for the unspliced (US), singly spliced (SS), and multiply spliced (MS) forms of HIV using specific primers. RNA fold change was calculated relative to DMSO treatment. (B) 293T cells were infected with HIV Δ env for 24 h in the presence of DMSO or sudemycin D6. Western blots were performed on cell lysates for HIV Gag products. Three experiments were quantitated (C). (D) 293T cells were infected with HIV Δ env for 24 h in the presence of DMSO or sudemycin D6. Western blots were performed on cell lysates for Tat and Rev. Data indicate means, and error bars indicate ± SEM ( n = 3). **, P

Techniques Used: Inhibition, Infection, Quantitative RT-PCR, Mass Spectrometry, Western Blot

Inhibition of SF3B1 prevents HIV reactivation from latency. (A) JLAT10.6 cells were incubated with the indicated compounds for 24 h, and FACS was performed to detect live GFP-positive cells as a measure of HIV reactivation from latency. (B) JLAT10.6 cells were incubated with the indicated compounds for 24 h. Cells were lysed and resolved on Western blots for the HIV products. A representative blot of 3 independent experiments is shown. (C) qRT-PCR for a similar experiment in panel B. Fold change is relative to DMSO treatment. (D) Inhibition of HIV reactivation in the Greene model of latency. Resting CD4 + T cells were infected with HIV-1 Luc, treated with protease inhibitor darunavir for 2 days. Cells were incubated with the compounds shown for 24 h in the presence of raltegravir. (See Materials and Methods for details.) HIV reactivation was measured by luciferase-based luminescence in the cell lysates normalized to protein concentration. (E) JLAT10.6 cells were treated with 5 μM sudemycin D6 for 18 h. Cells were washed and allowed to recover for 24 h. At 24, 48, or 72 h after drug washout, the cells were treated with LRAs for 24 h. HIV reactivation was measured with FACS to detect live cells expressing GFP. Data indicate means, and error bars indicate ±SEM ( n = 3). **, P
Figure Legend Snippet: Inhibition of SF3B1 prevents HIV reactivation from latency. (A) JLAT10.6 cells were incubated with the indicated compounds for 24 h, and FACS was performed to detect live GFP-positive cells as a measure of HIV reactivation from latency. (B) JLAT10.6 cells were incubated with the indicated compounds for 24 h. Cells were lysed and resolved on Western blots for the HIV products. A representative blot of 3 independent experiments is shown. (C) qRT-PCR for a similar experiment in panel B. Fold change is relative to DMSO treatment. (D) Inhibition of HIV reactivation in the Greene model of latency. Resting CD4 + T cells were infected with HIV-1 Luc, treated with protease inhibitor darunavir for 2 days. Cells were incubated with the compounds shown for 24 h in the presence of raltegravir. (See Materials and Methods for details.) HIV reactivation was measured by luciferase-based luminescence in the cell lysates normalized to protein concentration. (E) JLAT10.6 cells were treated with 5 μM sudemycin D6 for 18 h. Cells were washed and allowed to recover for 24 h. At 24, 48, or 72 h after drug washout, the cells were treated with LRAs for 24 h. HIV reactivation was measured with FACS to detect live cells expressing GFP. Data indicate means, and error bars indicate ±SEM ( n = 3). **, P

Techniques Used: Inhibition, Incubation, FACS, Western Blot, Quantitative RT-PCR, Infection, Protease Inhibitor, Luciferase, Protein Concentration, Expressing

21) Product Images from "Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements"

Article Title: Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements

Journal: Mutagenesis

doi: 10.1093/mutage/geq048

Schematic diagrams illustrating in vivo rearrangements of λgt10- lacZ transgene. Panels ( A ) through ( E ) show examples of rearranged λgt10- lacZ copies (R1–R5) that correspond to the rearrangement fragments of
Figure Legend Snippet: Schematic diagrams illustrating in vivo rearrangements of λgt10- lacZ transgene. Panels ( A ) through ( E ) show examples of rearranged λgt10- lacZ copies (R1–R5) that correspond to the rearrangement fragments of

Techniques Used: In Vivo

Summary of RT–PCR analysis of in vivo transgene copy number. Standard curves were based on quantifications of the ori amplicons, using replicated sets of template composed of CD2F 1 non-transgenic mouse DNA mixed with λgt10- lacZ (see Figure 3 Materials and methods) to generate haploid standards of 1, 2.5, 5, 10, 20, 40 and 50 copies. Cycle thresholds ( C t ) and efficiency parameters were obtained by iCycler software and show a linear correlation with log copy number values ( R 2 = 0.9924). Shown are average C t values for: ori standards (open circles), single-copy annexin V exon 4 (closed triangle, A); R5 rearrangement (closed circle, R5); interpolated copies of Muta™mouse ori (closed diamond, MM) and 18S (closed box, 18S).
Figure Legend Snippet: Summary of RT–PCR analysis of in vivo transgene copy number. Standard curves were based on quantifications of the ori amplicons, using replicated sets of template composed of CD2F 1 non-transgenic mouse DNA mixed with λgt10- lacZ (see Figure 3 Materials and methods) to generate haploid standards of 1, 2.5, 5, 10, 20, 40 and 50 copies. Cycle thresholds ( C t ) and efficiency parameters were obtained by iCycler software and show a linear correlation with log copy number values ( R 2 = 0.9924). Shown are average C t values for: ori standards (open circles), single-copy annexin V exon 4 (closed triangle, A); R5 rearrangement (closed circle, R5); interpolated copies of Muta™mouse ori (closed diamond, MM) and 18S (closed box, 18S).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vivo, Transgenic Assay, Software

Model of the Muta™Mouse λgt10- lacZ transgene derived by sequence analysis. The transgene monomer has 47 513 bp and 57 ORFs and is based on nucleotide sequencing of PCR amplicons derived from systematic scanning of functional regions of λgt10- lacZ using Muta™Mouse genomic DNA from tissues of both gender and λgt10- lacZ in vivo copies rescued by in vitro phage packaging and commercial stocks of λCI857 and λgt10. Data include the b527 deletion and unfinished parts of bacteriophage imm434 (accession numbers M60848, Y00118) and the 5.2 kb EcoR1-DraI fragment of pMC1511, which contains the E.coli lacZ mutation reporter (GenBank® L08935) (hatched-box). The GenBank® accession numbers for sequences other than those reported already for λ (NC_001416; 48.5 kb) are given in Table II . Functional regions included left (COS L ) and right (COS R ) cohesive ends and ORFs required for virion assembly and DNA packaging and origin of λ DNA replication (ORI). Arrows show orientation of ORFs common to the 48.5 kb λ bacteriophage as identified by λ gene nomenclature (see NC_001416). Novel in vivo copy rearrangements are described in Figure 2 and Table II . The large open box spans a novel finding of a region of substitution by lambdoid Rac prophage tail fiber assembly gene ( lom and ORFs 401, 314 and 194) found also in λgt10 and λCI857 commercial stocks. Also identified are crossover hot spot instigator motifs (Chi sites) conveying potential for lacZ recombination with the E.coli genome. The symbol χ + signifies a fully functional Chi motif (starts at nucleotide position 3508 on the reverse or antistrand with respect to E.coli lacZ (accession J01636). χ 0 signifies a one base difference version of the Chi motif that requires a single mutation for full function. These χ 0 sites start at nucleotide positions 3149 and 3152 for the two on the anti-strand and position 3248 for the one on the sense strand. MAR-1 and MAR-2 show location of sequences identified as matrix-associated regions using a computational approach ( 15 ). MAR-1 is located 5′ to the lambda INT gene and MAR-2 is located in the 3′ non-coding λgt-10 region and they represent regions that may contribute to higher order mammalian chromosome loop structures. In a head-to-tail arrangement of transgenes, MAR-2 would be located within 1.4 kb of the COS L site and in the vicinity of rearrangements featured in Figure 2 .
Figure Legend Snippet: Model of the Muta™Mouse λgt10- lacZ transgene derived by sequence analysis. The transgene monomer has 47 513 bp and 57 ORFs and is based on nucleotide sequencing of PCR amplicons derived from systematic scanning of functional regions of λgt10- lacZ using Muta™Mouse genomic DNA from tissues of both gender and λgt10- lacZ in vivo copies rescued by in vitro phage packaging and commercial stocks of λCI857 and λgt10. Data include the b527 deletion and unfinished parts of bacteriophage imm434 (accession numbers M60848, Y00118) and the 5.2 kb EcoR1-DraI fragment of pMC1511, which contains the E.coli lacZ mutation reporter (GenBank® L08935) (hatched-box). The GenBank® accession numbers for sequences other than those reported already for λ (NC_001416; 48.5 kb) are given in Table II . Functional regions included left (COS L ) and right (COS R ) cohesive ends and ORFs required for virion assembly and DNA packaging and origin of λ DNA replication (ORI). Arrows show orientation of ORFs common to the 48.5 kb λ bacteriophage as identified by λ gene nomenclature (see NC_001416). Novel in vivo copy rearrangements are described in Figure 2 and Table II . The large open box spans a novel finding of a region of substitution by lambdoid Rac prophage tail fiber assembly gene ( lom and ORFs 401, 314 and 194) found also in λgt10 and λCI857 commercial stocks. Also identified are crossover hot spot instigator motifs (Chi sites) conveying potential for lacZ recombination with the E.coli genome. The symbol χ + signifies a fully functional Chi motif (starts at nucleotide position 3508 on the reverse or antistrand with respect to E.coli lacZ (accession J01636). χ 0 signifies a one base difference version of the Chi motif that requires a single mutation for full function. These χ 0 sites start at nucleotide positions 3149 and 3152 for the two on the anti-strand and position 3248 for the one on the sense strand. MAR-1 and MAR-2 show location of sequences identified as matrix-associated regions using a computational approach ( 15 ). MAR-1 is located 5′ to the lambda INT gene and MAR-2 is located in the 3′ non-coding λgt-10 region and they represent regions that may contribute to higher order mammalian chromosome loop structures. In a head-to-tail arrangement of transgenes, MAR-2 would be located within 1.4 kb of the COS L site and in the vicinity of rearrangements featured in Figure 2 .

Techniques Used: Derivative Assay, Sequencing, Polymerase Chain Reaction, Functional Assay, In Vivo, In Vitro, Mutagenesis

22) Product Images from "Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach"

Article Title: Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach

Journal: PLoS ONE

doi: 10.1371/journal.pone.0139074

Four different KRAS multiplex digital PCR assays combining G12C, G12V and G12D mutant assays and corresponding duplex assays. KRAS G12C mutant droplet populations are indicated by a red dashed square, KRAS G12D mutant populations by a blue dashed square and KRAS G12V mutant populations by a yellow dashed square. Each multiplex assay, combining all relevant FAM and HEX assays, KRAS WT gDNA and G12V, D and C mutant gDNA, is shown in the top panel. The corresponding duplex assay for each mutation, using the same FAM and HEX assay concentration with KRAS WT DNA and the appropriate KRAS mutant DNA present, is shown in the panels below each multiplex assay. Multiplex 1 (top left panel) is an assay combination of 900 nM primers and 500 nM G12C probe, 562.5 nM primers and 312.5 nM G12D probe and 225 nM primers and 125 nM G12V probe with 450 nM primers and 250 nM WT for G12C probe. Multiplex 2 uses the same concentration of G12V and WT for G12C assay as Multiplex 1 but also contains 450 nM primers and 250 nM G12C probe and 675 nM primers and 375 nM G12D probe. Multiplex 3 uses the same concentration of KRAS mutant assays as in Multiplex 2 but with the WT for G12V probe assay present, used at the same concentration as the WT for G12C assay in Multiplex 1. Multiplex 4 is an assay combination of the G12C assay as in Multiplex 2 with 225 nM primers and 125 nM G12D probe and 675 nM primers and 375 nM G12V probe with the WT for G12D assay at the same concentration as the WT for G12C assay in Multiplex 1. Key: black- empty droplets, blue- mutant DNA FAM positive droplets, green- wild-type DNA HEX positive droplets, brown—wild-type and mutant DNA double positive droplets. The same scale is used for all panels (HEX amplitude up to 7000 and FAM amplitude up to 22000).
Figure Legend Snippet: Four different KRAS multiplex digital PCR assays combining G12C, G12V and G12D mutant assays and corresponding duplex assays. KRAS G12C mutant droplet populations are indicated by a red dashed square, KRAS G12D mutant populations by a blue dashed square and KRAS G12V mutant populations by a yellow dashed square. Each multiplex assay, combining all relevant FAM and HEX assays, KRAS WT gDNA and G12V, D and C mutant gDNA, is shown in the top panel. The corresponding duplex assay for each mutation, using the same FAM and HEX assay concentration with KRAS WT DNA and the appropriate KRAS mutant DNA present, is shown in the panels below each multiplex assay. Multiplex 1 (top left panel) is an assay combination of 900 nM primers and 500 nM G12C probe, 562.5 nM primers and 312.5 nM G12D probe and 225 nM primers and 125 nM G12V probe with 450 nM primers and 250 nM WT for G12C probe. Multiplex 2 uses the same concentration of G12V and WT for G12C assay as Multiplex 1 but also contains 450 nM primers and 250 nM G12C probe and 675 nM primers and 375 nM G12D probe. Multiplex 3 uses the same concentration of KRAS mutant assays as in Multiplex 2 but with the WT for G12V probe assay present, used at the same concentration as the WT for G12C assay in Multiplex 1. Multiplex 4 is an assay combination of the G12C assay as in Multiplex 2 with 225 nM primers and 125 nM G12D probe and 675 nM primers and 375 nM G12V probe with the WT for G12D assay at the same concentration as the WT for G12C assay in Multiplex 1. Key: black- empty droplets, blue- mutant DNA FAM positive droplets, green- wild-type DNA HEX positive droplets, brown—wild-type and mutant DNA double positive droplets. The same scale is used for all panels (HEX amplitude up to 7000 and FAM amplitude up to 22000).

Techniques Used: Multiplex Assay, Digital PCR, Mutagenesis, Concentration Assay

Correlation of NGS KRAS mutant allele frequency with digital PCR KRAS mutant allele frequency detected in the appropriate multiplex assay for FFPE tissue DNA and cell line gDNA samples.
Figure Legend Snippet: Correlation of NGS KRAS mutant allele frequency with digital PCR KRAS mutant allele frequency detected in the appropriate multiplex assay for FFPE tissue DNA and cell line gDNA samples.

Techniques Used: Next-Generation Sequencing, Mutagenesis, Digital PCR, Multiplex Assay, Formalin-fixed Paraffin-Embedded

KRAS multiplex digital PCR assays A-C and corresponding duplex assays. Multiplex A (top left panel) is an assay combination of 900 nM primers and 500 nM G13C probe (red dashed square), 450 nM primers and 250 nM G12C probe (blue dashed square) and 225 nM primers and 125 nM G12V probe (yellow dashed square). Multiplex B (top middle panel) is an assay combination of 675 nM primers and 375 nM G12S probe (red dashed square), 450 nM primers and 250 nM G12D probe (blue dashed square) and 225 nM primers and 125 nM G13D probe (yellow dashed square). Multiplex C (top right panel) is an assay combination of 675 nM primers and 375 nM G12R probe (red dashed square), 450 nM primers and 250 nM G12A probe (blue dashed square) and 900 nM primers and 500 nM Q61H probe (yellow dashed square). Multiplex C has 900 nM primers and 500 nM Q61H wild-type probe in addition to a G12C wild-type assay. All other wild-type droplet populations shown, except in the Q61H duplex assay, are 450 nM primers and 250 nM G12C wild-type probe. All panels in the left and centre columns show a FAM amplitude up to 18000 and an HEX amplitude up to 6000. Panels in the right column have a FAM amplitude up to 18000 and a HEX amplitude up to 11000.
Figure Legend Snippet: KRAS multiplex digital PCR assays A-C and corresponding duplex assays. Multiplex A (top left panel) is an assay combination of 900 nM primers and 500 nM G13C probe (red dashed square), 450 nM primers and 250 nM G12C probe (blue dashed square) and 225 nM primers and 125 nM G12V probe (yellow dashed square). Multiplex B (top middle panel) is an assay combination of 675 nM primers and 375 nM G12S probe (red dashed square), 450 nM primers and 250 nM G12D probe (blue dashed square) and 225 nM primers and 125 nM G13D probe (yellow dashed square). Multiplex C (top right panel) is an assay combination of 675 nM primers and 375 nM G12R probe (red dashed square), 450 nM primers and 250 nM G12A probe (blue dashed square) and 900 nM primers and 500 nM Q61H probe (yellow dashed square). Multiplex C has 900 nM primers and 500 nM Q61H wild-type probe in addition to a G12C wild-type assay. All other wild-type droplet populations shown, except in the Q61H duplex assay, are 450 nM primers and 250 nM G12C wild-type probe. All panels in the left and centre columns show a FAM amplitude up to 18000 and an HEX amplitude up to 6000. Panels in the right column have a FAM amplitude up to 18000 and a HEX amplitude up to 11000.

Techniques Used: Multiplex Assay, Digital PCR

23) Product Images from "A carotenogenic mini-pathway introduced into white corn does not affect development or agronomic performance"

Article Title: A carotenogenic mini-pathway introduced into white corn does not affect development or agronomic performance

Journal: Scientific Reports

doi: 10.1038/srep38288

Quantitative real-time RT-PCR analysis of endogenous carotenogenic genes and transgenes in Carolight® plants grown in the field (HC-F) or in the greenhouse (HC-GH). Data show relative mRNA levels in the immature endosperm of HC-F and HC-GH plants at four developmental stages (15, 20, 25 and 30 DAP) normalized against corn actin mRNA and presented as the mean of 10 biological replicates. ( a ) Zmbch1 , β-carotene hydroxylase 1; ( b ) Zmbch2 , β-carotene hydroxylase 2; ( c ) Zmcrtiso, carotene isomerase; ( d ) Zmcyp97a , carotene ε-hydroxylase; ( e ) Zmcyp97b ; ( f ) Zmcyp97c ; ( g ) Zmlycb , lycopene β-cyclase; ( h ) Zmlyce , lycopene ε-cyclase; ( i ) Zmpds , phytoene desaturase; ( j ) Zmzds , ζ-carotene desaturase; ( k ) Pacrti , phytoene desaturase; ( l ) Zmpsy1, phytoene synthase . Zm, Zea mays; Pa, Pantoea ananatis.
Figure Legend Snippet: Quantitative real-time RT-PCR analysis of endogenous carotenogenic genes and transgenes in Carolight® plants grown in the field (HC-F) or in the greenhouse (HC-GH). Data show relative mRNA levels in the immature endosperm of HC-F and HC-GH plants at four developmental stages (15, 20, 25 and 30 DAP) normalized against corn actin mRNA and presented as the mean of 10 biological replicates. ( a ) Zmbch1 , β-carotene hydroxylase 1; ( b ) Zmbch2 , β-carotene hydroxylase 2; ( c ) Zmcrtiso, carotene isomerase; ( d ) Zmcyp97a , carotene ε-hydroxylase; ( e ) Zmcyp97b ; ( f ) Zmcyp97c ; ( g ) Zmlycb , lycopene β-cyclase; ( h ) Zmlyce , lycopene ε-cyclase; ( i ) Zmpds , phytoene desaturase; ( j ) Zmzds , ζ-carotene desaturase; ( k ) Pacrti , phytoene desaturase; ( l ) Zmpsy1, phytoene synthase . Zm, Zea mays; Pa, Pantoea ananatis.

Techniques Used: Quantitative RT-PCR

24) Product Images from "Low-Level Parasite Persistence Drives Vasculitis and Myositis in Skeletal Muscle of Mice Chronically Infected with Trypanosoma cruzi"

Article Title: Low-Level Parasite Persistence Drives Vasculitis and Myositis in Skeletal Muscle of Mice Chronically Infected with Trypanosoma cruzi

Journal: Infection and Immunity

doi: 10.1128/IAI.00081-19

Benznidazole treatment during the chronic phase of T. cruzi infection strongly suppresses but does not eradicate the parasite. Female C57BL/6 mice were injected subcutaneously with 10 3 blood-stage trypomastigotes at 7 to 11 months of age and treated as shown. (A) Treatment protocols. The treatment schedules are indicated in Materials and Methods. (B to E) Treatment with benznidazole (Bz) from 2 to 4 months p.i. is defined as early benznidazole treatment (light green symbols in panels B to D); treatment with benznidazole from 9 to 11 months p.i. is defined as late benznidazole treatment (dark green symbols in panels B to D). Blue symbols in panels B to D denote animals receiving no treatment. The T. cruzi burden was determined quantitatively by counting clusters (B) and by DNA qPCR (C and D) as well as qualitatively by injecting 100 μl of blood from each mouse into Rag1 −/− mice (E). The number of Rag1 −/− mice that survived 60 days is given per the number of mice injected for each group. Each data point in panels B to D represents the parasitism of quadriceps muscle calculated for an individual mouse. Data for panels B to D are summarized from four independent experiments. Differences between cohorts in panels B to D were compared using two-tailed Student’s t test. Differences between cohorts in panel E were compared using Fisher’s exact test (ns, not significant [ P   >  0.05]; *, 0.05 ≥  P   >  5 × 10 −4 ; **, 5 × 10 −4 ≥ P   >  5 × 10 −6 ; ***, P  
Figure Legend Snippet: Benznidazole treatment during the chronic phase of T. cruzi infection strongly suppresses but does not eradicate the parasite. Female C57BL/6 mice were injected subcutaneously with 10 3 blood-stage trypomastigotes at 7 to 11 months of age and treated as shown. (A) Treatment protocols. The treatment schedules are indicated in Materials and Methods. (B to E) Treatment with benznidazole (Bz) from 2 to 4 months p.i. is defined as early benznidazole treatment (light green symbols in panels B to D); treatment with benznidazole from 9 to 11 months p.i. is defined as late benznidazole treatment (dark green symbols in panels B to D). Blue symbols in panels B to D denote animals receiving no treatment. The T. cruzi burden was determined quantitatively by counting clusters (B) and by DNA qPCR (C and D) as well as qualitatively by injecting 100 μl of blood from each mouse into Rag1 −/− mice (E). The number of Rag1 −/− mice that survived 60 days is given per the number of mice injected for each group. Each data point in panels B to D represents the parasitism of quadriceps muscle calculated for an individual mouse. Data for panels B to D are summarized from four independent experiments. Differences between cohorts in panels B to D were compared using two-tailed Student’s t test. Differences between cohorts in panel E were compared using Fisher’s exact test (ns, not significant [ P   >  0.05]; *, 0.05 ≥  P   >  5 × 10 −4 ; **, 5 × 10 −4 ≥ P   >  5 × 10 −6 ; ***, P  

Techniques Used: Infection, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, Two Tailed Test

25) Product Images from "Contributions of CTCF and DNA Methyltransferases DNMT1 and DNMT3B to Epstein-Barr Virus Restricted Latency"

Article Title: Contributions of CTCF and DNA Methyltransferases DNMT1 and DNMT3B to Epstein-Barr Virus Restricted Latency

Journal: Journal of Virology

doi: 10.1128/JVI.05923-11

Reduction of DNMT3B levels in BL cells maintaining latency I does not result in reactivation of the latency III program. (A) qRT-PCR analysis of DNMT3B in Kem I (left panel) and Mutu I (right panel) BL cell lines stably expressing control (Ctl.) or DNMT3B-specific (shDNMT3B) shRNAs. (B) EBV EBNA2 and LMP1 expression was assessed as markers of potential reactivation of the latency III program in the cell lines analyzed in panel A. Immunoblot detection of β-actin served as a loading control. The one-sample Student t test was used to determine statistical differences.
Figure Legend Snippet: Reduction of DNMT3B levels in BL cells maintaining latency I does not result in reactivation of the latency III program. (A) qRT-PCR analysis of DNMT3B in Kem I (left panel) and Mutu I (right panel) BL cell lines stably expressing control (Ctl.) or DNMT3B-specific (shDNMT3B) shRNAs. (B) EBV EBNA2 and LMP1 expression was assessed as markers of potential reactivation of the latency III program in the cell lines analyzed in panel A. Immunoblot detection of β-actin served as a loading control. The one-sample Student t test was used to determine statistical differences.

Techniques Used: Quantitative RT-PCR, Stable Transfection, Expressing, CTL Assay

26) Product Images from "Extension of the Germinal Center Stage of B-cell Development Promotes Autoantibodies in BXD2 Mice"

Article Title: Extension of the Germinal Center Stage of B-cell Development Promotes Autoantibodies in BXD2 Mice

Journal: Arthritis and rheumatism

doi: 10.1002/art.38059

Decreased GC program and increased plasma program gene expression in the absence of RGS13. qRT-PCR analysis of ( A ) GC program-related, ( B ) Plasma program-related, and ( C ) pCREB targeted or POU family transcription factor genes (CD19 + PNA + Fas + ) sorted from
Figure Legend Snippet: Decreased GC program and increased plasma program gene expression in the absence of RGS13. qRT-PCR analysis of ( A ) GC program-related, ( B ) Plasma program-related, and ( C ) pCREB targeted or POU family transcription factor genes (CD19 + PNA + Fas + ) sorted from

Techniques Used: Expressing, Quantitative RT-PCR

Induction of Rgs13 in GC B cells by IL-17. A , qRT-PCR analysis of Rgs13 expression in B cells sorted from the spleens of indicated strains (ND = not detectable; ** p
Figure Legend Snippet: Induction of Rgs13 in GC B cells by IL-17. A , qRT-PCR analysis of Rgs13 expression in B cells sorted from the spleens of indicated strains (ND = not detectable; ** p

Techniques Used: Quantitative RT-PCR, Expressing

27) Product Images from "Ascorbyl stearate stimulates cell death by oxidative stress-mediated apoptosis and autophagy in HeLa cervical cancer cell line in vitro"

Article Title: Ascorbyl stearate stimulates cell death by oxidative stress-mediated apoptosis and autophagy in HeLa cervical cancer cell line in vitro

Journal: 3 Biotech

doi: 10.1007/s13205-019-1628-5

Nrf-2 levels in HeLa cells were probed by EMSA and RT-qPCR. EMSA profile of Nrfr-2 a presence and absence of Asc-s at concentration dependent for 6 h incubation, b time-dependent manner when treated with 100 µM Asc-s, c, d dose-dependent decrease in qRT-PCR profile of Nrf-2 mRNA levels in HeLa cell lysate treated with Asc-s (0–200 µM) for 6 h and in HeLa cell lysate treated with 126 µM Asc-s for 0–6 h, when compared with untreated HeLa cells. Data represent mean ± SE ( n = 3). Values are significant at * p
Figure Legend Snippet: Nrf-2 levels in HeLa cells were probed by EMSA and RT-qPCR. EMSA profile of Nrfr-2 a presence and absence of Asc-s at concentration dependent for 6 h incubation, b time-dependent manner when treated with 100 µM Asc-s, c, d dose-dependent decrease in qRT-PCR profile of Nrf-2 mRNA levels in HeLa cell lysate treated with Asc-s (0–200 µM) for 6 h and in HeLa cell lysate treated with 126 µM Asc-s for 0–6 h, when compared with untreated HeLa cells. Data represent mean ± SE ( n = 3). Values are significant at * p

Techniques Used: Quantitative RT-PCR, Concentration Assay, Incubation

Effect of Asc-s on murine splenic lymphocytes. a – e Histogram depicting the effect of different concentrations of Asc-s on murine splenic lymphocytes investigated by propodium iodide staining and analysed by high content screening instrument (Acumen Celista) from TTP Labtech, UK. f Bar diagram illustrates significant data point mean ± SE of three independent experiments. No cell death of splenic lymphocytes on treatment with different concentrations of Asc-s
Figure Legend Snippet: Effect of Asc-s on murine splenic lymphocytes. a – e Histogram depicting the effect of different concentrations of Asc-s on murine splenic lymphocytes investigated by propodium iodide staining and analysed by high content screening instrument (Acumen Celista) from TTP Labtech, UK. f Bar diagram illustrates significant data point mean ± SE of three independent experiments. No cell death of splenic lymphocytes on treatment with different concentrations of Asc-s

Techniques Used: Staining, High Content Screening

Immunoblot and real-time PCR profile of autophagy protein LC3. a HeLa cells were treated with Asc-s (0–200 µM) for 48 h. Cell lysates were processed for immunoblot study to detect LC3 and β-actin as loading control. b Dose-dependent decrease in LC3-I levels was observed in HeLa cell lysate treated with Asc-s (0–200 µM) for 48 h when compared with untreated HeLa cells, c dose-dependent increase in LC3-II levels in HeLa cell lysate treated with Asc-s (0–200 µM) for 48 h when compared with untreated HeLa cells, d dose-dependent increase in RT-qPCR profile of LC3 mRNA levels in HeLa cell lysate treated with Asc-s (0–200 µM) for 48 h when compared with untreated HeLa cells. Data represent mean ± SE ( n = 3). Values are significant at * p
Figure Legend Snippet: Immunoblot and real-time PCR profile of autophagy protein LC3. a HeLa cells were treated with Asc-s (0–200 µM) for 48 h. Cell lysates were processed for immunoblot study to detect LC3 and β-actin as loading control. b Dose-dependent decrease in LC3-I levels was observed in HeLa cell lysate treated with Asc-s (0–200 µM) for 48 h when compared with untreated HeLa cells, c dose-dependent increase in LC3-II levels in HeLa cell lysate treated with Asc-s (0–200 µM) for 48 h when compared with untreated HeLa cells, d dose-dependent increase in RT-qPCR profile of LC3 mRNA levels in HeLa cell lysate treated with Asc-s (0–200 µM) for 48 h when compared with untreated HeLa cells. Data represent mean ± SE ( n = 3). Values are significant at * p

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

a Effect of different concentrations of Asc-s on viability of HeLa cells determined by MTT assay. A dose-dependent decrease in cell viability was observed on treatment with Asc-s. b, c Percent daughter cells of HeLa estimated by CFSE dye method at different concentrations of Asc-s treatment. d – h CFSE profile of HeLa cells treated with 0, 50, 100, 150, and 200 µM Asc-s for 48 h. A significant dose-dependent decrease in CFSE counts was observed in HeLa cells on treatment with 50, 100, 150, 200 µM Asc-s. Data represent mean ± SE ( n = 3). Values are significant at ** p
Figure Legend Snippet: a Effect of different concentrations of Asc-s on viability of HeLa cells determined by MTT assay. A dose-dependent decrease in cell viability was observed on treatment with Asc-s. b, c Percent daughter cells of HeLa estimated by CFSE dye method at different concentrations of Asc-s treatment. d – h CFSE profile of HeLa cells treated with 0, 50, 100, 150, and 200 µM Asc-s for 48 h. A significant dose-dependent decrease in CFSE counts was observed in HeLa cells on treatment with 50, 100, 150, 200 µM Asc-s. Data represent mean ± SE ( n = 3). Values are significant at ** p

Techniques Used: MTT Assay

Effect of Asc-s on cell cycle distribution of HeLa cells. a – e Cells treated with various concentrations of Asc-s (0, 50, 100,150, and 200 µM) for 48 h were collected and stained with propodium iodide and analysed by flow cytometry. f Significant accumulation of cells at sub-G0/G1 at 50, 100, 150, and 200 µM of Asc-s treatment was observed compared with control cells. Data represent mean ± SE of three independent experiments; values are significant at **** p
Figure Legend Snippet: Effect of Asc-s on cell cycle distribution of HeLa cells. a – e Cells treated with various concentrations of Asc-s (0, 50, 100,150, and 200 µM) for 48 h were collected and stained with propodium iodide and analysed by flow cytometry. f Significant accumulation of cells at sub-G0/G1 at 50, 100, 150, and 200 µM of Asc-s treatment was observed compared with control cells. Data represent mean ± SE of three independent experiments; values are significant at **** p

Techniques Used: Staining, Flow Cytometry

Effect of Asc-s treatment on ROS levels in HeLa cells measured by DCFH2 method. Dose-dependent increase in ROS levels was observed at 15–60 min of 50–200 µM Asc-s treatment. Antioxidant control comprises of (Trolox, 100 µM) with 126 µM Asc-s treatment followed by DCF-DA staining. Data represent mean ± SE ( n = 3). Values are significant at ** p
Figure Legend Snippet: Effect of Asc-s treatment on ROS levels in HeLa cells measured by DCFH2 method. Dose-dependent increase in ROS levels was observed at 15–60 min of 50–200 µM Asc-s treatment. Antioxidant control comprises of (Trolox, 100 µM) with 126 µM Asc-s treatment followed by DCF-DA staining. Data represent mean ± SE ( n = 3). Values are significant at ** p

Techniques Used: Staining

Pro-apoptotic effect of Asc-s on HeLa cells. a DAPI-stained control HeLa cells without blue fluorescence when treated with DMSO for 48 h, b DAPI-stained HeLa cells treated with 126 µM Asc-s for 24 h, c DAPI-stained HeLa cells apoptotic bodies with bright blue fluorescence and more nuclear or chromatin condensation when treated with 126 µM Asc-s for 48 h, arrows indicate apoptotic bodies of nuclear fragmentation and chromatin condensation (4 B and 4 C, respectively), d, g PI and AO stained control HeLa cells treated with DMSO for 48 h, e, h PI and AO stained HeLa cells treated with 126 µM Asc-s for 24 h, showed early apoptosis features represented as intercalated acridine orange (bright green) amongst the fragmented DNA (arrows), f, i PI and AO stained HeLa cells treated with 126 µM of Asc-s for 48 h, depicts blebbing and nuclear margination noticeable at 48 h treatment (arrows)
Figure Legend Snippet: Pro-apoptotic effect of Asc-s on HeLa cells. a DAPI-stained control HeLa cells without blue fluorescence when treated with DMSO for 48 h, b DAPI-stained HeLa cells treated with 126 µM Asc-s for 24 h, c DAPI-stained HeLa cells apoptotic bodies with bright blue fluorescence and more nuclear or chromatin condensation when treated with 126 µM Asc-s for 48 h, arrows indicate apoptotic bodies of nuclear fragmentation and chromatin condensation (4 B and 4 C, respectively), d, g PI and AO stained control HeLa cells treated with DMSO for 48 h, e, h PI and AO stained HeLa cells treated with 126 µM Asc-s for 24 h, showed early apoptosis features represented as intercalated acridine orange (bright green) amongst the fragmented DNA (arrows), f, i PI and AO stained HeLa cells treated with 126 µM of Asc-s for 48 h, depicts blebbing and nuclear margination noticeable at 48 h treatment (arrows)

Techniques Used: Staining, Fluorescence

28) Product Images from "Vortex fluidics-mediated DNA rescue from formalin-fixed museum specimens"

Article Title: Vortex fluidics-mediated DNA rescue from formalin-fixed museum specimens

Journal: PLoS ONE

doi: 10.1371/journal.pone.0225807

Schematic of the VFD-mediated fDNA recovery technique. (A) The protocol begins with Vortex Fluidic Device (VFD) treatment (7 krpm, room temperature, abbreviated RT, 1 h) of a mixture of proteinase K and the frozen, then broken-up tissue. The reaction mixture is next processed to remove solids and DNA polymerase inhibitors. The recovered fDNA is then purified and concentrated. Finally, the DNA is amplified, quantified, and characterized by (B) qPCR and (C) DNA sequencing of the samples. Larger versions of panels B and C are provided in S1 and S2 Figs. Threshold cycle (C t ) and endpoint fluorescence values are given in S1 Table .
Figure Legend Snippet: Schematic of the VFD-mediated fDNA recovery technique. (A) The protocol begins with Vortex Fluidic Device (VFD) treatment (7 krpm, room temperature, abbreviated RT, 1 h) of a mixture of proteinase K and the frozen, then broken-up tissue. The reaction mixture is next processed to remove solids and DNA polymerase inhibitors. The recovered fDNA is then purified and concentrated. Finally, the DNA is amplified, quantified, and characterized by (B) qPCR and (C) DNA sequencing of the samples. Larger versions of panels B and C are provided in S1 and S2 Figs. Threshold cycle (C t ) and endpoint fluorescence values are given in S1 Table .

Techniques Used: Purification, Amplification, Real-time Polymerase Chain Reaction, DNA Sequencing, Fluorescence

Amplification of an 183-bp fDNA target from the ATP synthase gene of the lobster mitochondrial genome. (A) Quantitative PCR and (B) agarose DNA gel electrophoresis identified 7 krpm as the optimal VFD rotational speed for qPCR amplification. Threshold cycle and endpoint fluorescence values are provided in S2 Table . The variable-rotational speed PCR reactions were compared to a no template control (NTC), a fresh lobster DNA positive control (+), and a non-VFD-processed negative control (–). (C) The 7 krpm VFD-processed qPCR product (*) was subjected to Sanger sequencing; a mutation (G2728A, GenBank No. HQ402925) was observed (highlighted).
Figure Legend Snippet: Amplification of an 183-bp fDNA target from the ATP synthase gene of the lobster mitochondrial genome. (A) Quantitative PCR and (B) agarose DNA gel electrophoresis identified 7 krpm as the optimal VFD rotational speed for qPCR amplification. Threshold cycle and endpoint fluorescence values are provided in S2 Table . The variable-rotational speed PCR reactions were compared to a no template control (NTC), a fresh lobster DNA positive control (+), and a non-VFD-processed negative control (–). (C) The 7 krpm VFD-processed qPCR product (*) was subjected to Sanger sequencing; a mutation (G2728A, GenBank No. HQ402925) was observed (highlighted).

Techniques Used: Amplification, Real-time Polymerase Chain Reaction, DNA Gel Electrophoresis, Fluorescence, Polymerase Chain Reaction, Positive Control, Negative Control, Sequencing, Mutagenesis

29) Product Images from "Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri"

Article Title: Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri

Journal: PLoS ONE

doi: 10.1371/journal.pone.0159004

Calibration curves of qPCR assays run with positive plasmid DNA (unbroken line) and bacterial suspension (broken line). Plasmid DNA was tenfold diluted serially from 5.88E+6–5.88E+0 copies/μL. The slope of the plasmid DNA standard curve is –3.3154, equivalent to an efficiency of 100.3% ( R 2 = 0.9993). The bacterial suspension was 10-fold serially diluted from 1.78E+8–1.78E+1 CFU/μL. The slope of the bacterial suspension calibration curve is –3.0369, equivalent to an efficiency of 113.5% ( R 2 = 0.9955), indicating PCR inhibition probably caused by the residual medium matrix.
Figure Legend Snippet: Calibration curves of qPCR assays run with positive plasmid DNA (unbroken line) and bacterial suspension (broken line). Plasmid DNA was tenfold diluted serially from 5.88E+6–5.88E+0 copies/μL. The slope of the plasmid DNA standard curve is –3.3154, equivalent to an efficiency of 100.3% ( R 2 = 0.9993). The bacterial suspension was 10-fold serially diluted from 1.78E+8–1.78E+1 CFU/μL. The slope of the bacterial suspension calibration curve is –3.0369, equivalent to an efficiency of 113.5% ( R 2 = 0.9955), indicating PCR inhibition probably caused by the residual medium matrix.

Techniques Used: Real-time Polymerase Chain Reaction, Plasmid Preparation, Polymerase Chain Reaction, Inhibition

30) Product Images from "Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements"

Article Title: Characterisation of Muta(TM)Mouse ?gt10-lacZ transgene: evidence for in vivo rearrangements

Journal: Mutagenesis

doi: 10.1093/mutage/geq048

Summary of RT–PCR analysis of in vivo transgene copy number. Standard curves were based on quantifications of the ori amplicons, using replicated sets of template composed of CD2F 1 non-transgenic mouse DNA mixed with λgt10- lacZ (see Figure 3 Materials and methods) to generate haploid standards of 1, 2.5, 5, 10, 20, 40 and 50 copies. Cycle thresholds ( C t ) and efficiency parameters were obtained by iCycler software and show a linear correlation with log copy number values ( R 2 = 0.9924). Shown are average C t values for: ori standards (open circles), single-copy annexin V exon 4 (closed triangle, A); R5 rearrangement (closed circle, R5); interpolated copies of Muta™mouse ori (closed diamond, MM) and 18S (closed box, 18S).
Figure Legend Snippet: Summary of RT–PCR analysis of in vivo transgene copy number. Standard curves were based on quantifications of the ori amplicons, using replicated sets of template composed of CD2F 1 non-transgenic mouse DNA mixed with λgt10- lacZ (see Figure 3 Materials and methods) to generate haploid standards of 1, 2.5, 5, 10, 20, 40 and 50 copies. Cycle thresholds ( C t ) and efficiency parameters were obtained by iCycler software and show a linear correlation with log copy number values ( R 2 = 0.9924). Shown are average C t values for: ori standards (open circles), single-copy annexin V exon 4 (closed triangle, A); R5 rearrangement (closed circle, R5); interpolated copies of Muta™mouse ori (closed diamond, MM) and 18S (closed box, 18S).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vivo, Transgenic Assay, Software

Model of the Muta™Mouse λgt10- lacZ transgene derived by sequence analysis. The transgene monomer has 47 513 bp and 57 ORFs and is based on nucleotide sequencing of PCR amplicons derived from systematic scanning of functional regions of λgt10- lacZ using Muta™Mouse genomic DNA from tissues of both gender and λgt10- lacZ in vivo copies rescued by in vitro phage packaging and commercial stocks of λCI857 and λgt10. Data include the b527 deletion and unfinished parts of bacteriophage imm434 (accession numbers M60848, Y00118) and the 5.2 kb EcoR1-DraI fragment of pMC1511, which contains the E.coli lacZ mutation reporter (GenBank® L08935) (hatched-box). The GenBank® accession numbers for sequences other than those reported already for λ (NC_001416; 48.5 kb) are given in Table II . Functional regions included left (COS L ) and right (COS R ) cohesive ends and ORFs required for virion assembly and DNA packaging and origin of λ DNA replication (ORI). Arrows show orientation of ORFs common to the 48.5 kb λ bacteriophage as identified by λ gene nomenclature (see NC_001416). Novel in vivo copy rearrangements are described in Figure 2 and Table II . The large open box spans a novel finding of a region of substitution by lambdoid Rac prophage tail fiber assembly gene ( lom and ORFs 401, 314 and 194) found also in λgt10 and λCI857 commercial stocks. Also identified are crossover hot spot instigator motifs (Chi sites) conveying potential for lacZ recombination with the E.coli genome. The symbol χ + signifies a fully functional Chi motif (starts at nucleotide position 3508 on the reverse or antistrand with respect to E.coli lacZ (accession J01636). χ 0 signifies a one base difference version of the Chi motif that requires a single mutation for full function. These χ 0 sites start at nucleotide positions 3149 and 3152 for the two on the anti-strand and position 3248 for the one on the sense strand. MAR-1 and MAR-2 show location of sequences identified as matrix-associated regions using a computational approach ( 15 ). MAR-1 is located 5′ to the lambda INT gene and MAR-2 is located in the 3′ non-coding λgt-10 region and they represent regions that may contribute to higher order mammalian chromosome loop structures. In a head-to-tail arrangement of transgenes, MAR-2 would be located within 1.4 kb of the COS L site and in the vicinity of rearrangements featured in Figure 2 .
Figure Legend Snippet: Model of the Muta™Mouse λgt10- lacZ transgene derived by sequence analysis. The transgene monomer has 47 513 bp and 57 ORFs and is based on nucleotide sequencing of PCR amplicons derived from systematic scanning of functional regions of λgt10- lacZ using Muta™Mouse genomic DNA from tissues of both gender and λgt10- lacZ in vivo copies rescued by in vitro phage packaging and commercial stocks of λCI857 and λgt10. Data include the b527 deletion and unfinished parts of bacteriophage imm434 (accession numbers M60848, Y00118) and the 5.2 kb EcoR1-DraI fragment of pMC1511, which contains the E.coli lacZ mutation reporter (GenBank® L08935) (hatched-box). The GenBank® accession numbers for sequences other than those reported already for λ (NC_001416; 48.5 kb) are given in Table II . Functional regions included left (COS L ) and right (COS R ) cohesive ends and ORFs required for virion assembly and DNA packaging and origin of λ DNA replication (ORI). Arrows show orientation of ORFs common to the 48.5 kb λ bacteriophage as identified by λ gene nomenclature (see NC_001416). Novel in vivo copy rearrangements are described in Figure 2 and Table II . The large open box spans a novel finding of a region of substitution by lambdoid Rac prophage tail fiber assembly gene ( lom and ORFs 401, 314 and 194) found also in λgt10 and λCI857 commercial stocks. Also identified are crossover hot spot instigator motifs (Chi sites) conveying potential for lacZ recombination with the E.coli genome. The symbol χ + signifies a fully functional Chi motif (starts at nucleotide position 3508 on the reverse or antistrand with respect to E.coli lacZ (accession J01636). χ 0 signifies a one base difference version of the Chi motif that requires a single mutation for full function. These χ 0 sites start at nucleotide positions 3149 and 3152 for the two on the anti-strand and position 3248 for the one on the sense strand. MAR-1 and MAR-2 show location of sequences identified as matrix-associated regions using a computational approach ( 15 ). MAR-1 is located 5′ to the lambda INT gene and MAR-2 is located in the 3′ non-coding λgt-10 region and they represent regions that may contribute to higher order mammalian chromosome loop structures. In a head-to-tail arrangement of transgenes, MAR-2 would be located within 1.4 kb of the COS L site and in the vicinity of rearrangements featured in Figure 2 .

Techniques Used: Derivative Assay, Sequencing, Polymerase Chain Reaction, Functional Assay, In Vivo, In Vitro, Mutagenesis

31) Product Images from "Identification of Claudin 1 Transcript Variants in Human Invasive Breast Cancer"

Article Title: Identification of Claudin 1 Transcript Variants in Human Invasive Breast Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0163387

Identification of CLDN1 transcript variants in invasive human breast cancer. PCR analysis was carried out on reverse transcribed RNA from 12 breast tumors using primers ( Table 1 ) flanking the coding regions of CLDN1 , 3 , and 4 . The expected full length cDNA products (representing the classical transcripts) for CLDN1 and CLDN4 (700 bases and 706 bases respectively) was evident in all tumors. However, no CLDN3 transcript was detected in some of the breast tumors (middle panel; lanes 4, 10, 11). Colored arrows indicate CLDN1 PCR products which were verified by Sanger sequencing: yellow arrow, transcript variant 1 (V1) is 615 bp; red arrow, transcript variant 2 (V2) is 440bp; blue arrow, transcript variant 3 (V3) is 362 bp; and green arrow, transcript variant 4 (V4) is 217 bp. In the lower panel, the product indicated by the black arrow, was a non-specific band and had no sequence homology to CLDN4 .
Figure Legend Snippet: Identification of CLDN1 transcript variants in invasive human breast cancer. PCR analysis was carried out on reverse transcribed RNA from 12 breast tumors using primers ( Table 1 ) flanking the coding regions of CLDN1 , 3 , and 4 . The expected full length cDNA products (representing the classical transcripts) for CLDN1 and CLDN4 (700 bases and 706 bases respectively) was evident in all tumors. However, no CLDN3 transcript was detected in some of the breast tumors (middle panel; lanes 4, 10, 11). Colored arrows indicate CLDN1 PCR products which were verified by Sanger sequencing: yellow arrow, transcript variant 1 (V1) is 615 bp; red arrow, transcript variant 2 (V2) is 440bp; blue arrow, transcript variant 3 (V3) is 362 bp; and green arrow, transcript variant 4 (V4) is 217 bp. In the lower panel, the product indicated by the black arrow, was a non-specific band and had no sequence homology to CLDN4 .

Techniques Used: Polymerase Chain Reaction, Sequencing, Variant Assay

32) Product Images from "Seasonal and Regional Dynamics of M. ulcerans Transmission in Environmental Context: Deciphering the Role of Water Bugs as Hosts and Vectors"

Article Title: Seasonal and Regional Dynamics of M. ulcerans Transmission in Environmental Context: Deciphering the Role of Water Bugs as Hosts and Vectors

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0000731

Detection of M. ulcerans DNA in insect tissues. The analysis was performed on 616 insect pools corresponding to 3647 individual specimens. (A) Percentage of positive pools over the period of study, (n) corresponding to the number of positive pools out of total pools. (B) Number of pools and number of positive pools (black part of bars represent the number of positive PCR pools) following water bug families and period of collection. (C) Monthly trends in M. ulcerans DNA positivity rate by family.
Figure Legend Snippet: Detection of M. ulcerans DNA in insect tissues. The analysis was performed on 616 insect pools corresponding to 3647 individual specimens. (A) Percentage of positive pools over the period of study, (n) corresponding to the number of positive pools out of total pools. (B) Number of pools and number of positive pools (black part of bars represent the number of positive PCR pools) following water bug families and period of collection. (C) Monthly trends in M. ulcerans DNA positivity rate by family.

Techniques Used: Polymerase Chain Reaction

33) Product Images from "Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani"

Article Title: Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani

Journal: Experimental parasitology

doi: 10.1016/j.exppara.2014.12.003

3.1. Sensitivities of Babesia real-time PCR and ddPCR assays
Figure Legend Snippet: 3.1. Sensitivities of Babesia real-time PCR and ddPCR assays

Techniques Used: Real-time Polymerase Chain Reaction

Quantification of Babesia microti ITS1 DNA in infected hamsters by real-time PCR (RT-PCR) and ddPCR. (A) DNA copy numbers measured by RT-PCR (black bars) and ddPCR (gray bars) were transformed to log 10 values. Non-transformed copy numbers are shown in
Figure Legend Snippet: Quantification of Babesia microti ITS1 DNA in infected hamsters by real-time PCR (RT-PCR) and ddPCR. (A) DNA copy numbers measured by RT-PCR (black bars) and ddPCR (gray bars) were transformed to log 10 values. Non-transformed copy numbers are shown in

Techniques Used: Infection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transformation Assay

3.1. Sensitivities of Babesia real-time PCR and ddPCR assays
Figure Legend Snippet: 3.1. Sensitivities of Babesia real-time PCR and ddPCR assays

Techniques Used: Real-time Polymerase Chain Reaction

34) Product Images from "Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice"

Article Title: Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice

Journal: Skeletal Muscle

doi: 10.1186/s13395-016-0075-9

Endurance exercise prevents dysregulated mitochondrial-induced apoptosis, reduces nuclear p53-mediated repression of PGC-1α, induces PGC-1α regulated gene networks, restores mtDNA copy number, and normalizes mitochondrial morphology in mtDNA mutator mice. a Nuclear DNA fragmentation (apoptotic index) in the heart, HSC, and SC of WT, PolG-SED, and PolG-END ( n = 7–10/group). b ChIP assay showing reduced p53 enrichment of PGC-1α promoter (positions −954/−564) with exercise in the muscle and heart of WT, PolG-SED, and PolG-END ( n = 6/group). c Gene expression of PGC-1α and its downstream targets in muscle ( quadriceps femoris ), d mtDNA copy number normalized to nuclear β-globin gene in the muscle ( soleus ) and heart, and e representative electron micrographs of myofibers ( quadriceps femoris ) and cardiomyocytes (heart) from WT, PolG-SED, and PolG-END mice ( n = 4–7/group). Asterisk (PolG-SED vs. both WT and PolG-END): * P
Figure Legend Snippet: Endurance exercise prevents dysregulated mitochondrial-induced apoptosis, reduces nuclear p53-mediated repression of PGC-1α, induces PGC-1α regulated gene networks, restores mtDNA copy number, and normalizes mitochondrial morphology in mtDNA mutator mice. a Nuclear DNA fragmentation (apoptotic index) in the heart, HSC, and SC of WT, PolG-SED, and PolG-END ( n = 7–10/group). b ChIP assay showing reduced p53 enrichment of PGC-1α promoter (positions −954/−564) with exercise in the muscle and heart of WT, PolG-SED, and PolG-END ( n = 6/group). c Gene expression of PGC-1α and its downstream targets in muscle ( quadriceps femoris ), d mtDNA copy number normalized to nuclear β-globin gene in the muscle ( soleus ) and heart, and e representative electron micrographs of myofibers ( quadriceps femoris ) and cardiomyocytes (heart) from WT, PolG-SED, and PolG-END mice ( n = 4–7/group). Asterisk (PolG-SED vs. both WT and PolG-END): * P

Techniques Used: Pyrolysis Gas Chromatography, Mouse Assay, Chromatin Immunoprecipitation, Expressing

Endurance exercise-mediated repair of mtDNA mutations is mitochondrial p53-dependent. a A fluorescence-based in vitro DNA primer extension-mutation repair assay in muscle mitochondrial extracts from WT, PolG-SED, and PolG-END ( n = 6–8/group) to assess the excision of the unpaired artificial point mutations. b p53 immunodepletion prevents mutation repair in muscle mitochondrial extracts from PolG-END ( n = 5/group). A non-specific IgG antibody was used as negative control antibody. c Endurance stress test time to exhaustion in four independent trials in WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice ( n = 5–6/group). d Representative electron micrographs of myofibers ( quadriceps femoris ) from WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END ( n = 4/group). e Random mtDNA somatic mutation rate (per 1000 nucleotides of mtDNA) in muscle ( quadriceps femoris ) WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice ( n = 3–4/group). f mtDNA copy number in muscle mitochondria from PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice ( n = 4–5/group) relative to WT mice ( horizontal line ). g Cytochrome c oxidase (COX) activity in muscle from WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice ( n = 4–5/group). Asterisk (PolG-SED vs. both WT and PolG-END): * P
Figure Legend Snippet: Endurance exercise-mediated repair of mtDNA mutations is mitochondrial p53-dependent. a A fluorescence-based in vitro DNA primer extension-mutation repair assay in muscle mitochondrial extracts from WT, PolG-SED, and PolG-END ( n = 6–8/group) to assess the excision of the unpaired artificial point mutations. b p53 immunodepletion prevents mutation repair in muscle mitochondrial extracts from PolG-END ( n = 5/group). A non-specific IgG antibody was used as negative control antibody. c Endurance stress test time to exhaustion in four independent trials in WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice ( n = 5–6/group). d Representative electron micrographs of myofibers ( quadriceps femoris ) from WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END ( n = 4/group). e Random mtDNA somatic mutation rate (per 1000 nucleotides of mtDNA) in muscle ( quadriceps femoris ) WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice ( n = 3–4/group). f mtDNA copy number in muscle mitochondria from PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice ( n = 4–5/group) relative to WT mice ( horizontal line ). g Cytochrome c oxidase (COX) activity in muscle from WT, PolG-SED, PolG-END, PolG-p53 MKO-SED, and PolG-p53 MKO-END mice ( n = 4–5/group). Asterisk (PolG-SED vs. both WT and PolG-END): * P

Techniques Used: Fluorescence, In Vitro, Mutagenesis, Negative Control, Mouse Assay, Activity Assay

Endurance exercise increases the abundance of p53 in mitochondrial matrix where it interacts with mtDNA in a complex with POLG1 and Tfam in mtDNA mutator mice. a Mitochondrial p53 is primarily localized in the matrix. Muscle mitochondria were subfractionated into outer mitochondrial membrane (OMM), intermembrane space (IMS), inner mitochondrial membrane (IMM), and matrix (Mx) fractions, and these fractions were immunoblotted for the compartment-specific proteins TOMM22 (~16 kDa), cytochrome c (~14 kDa), COX-IV (~17 kDa), and CS (~45 kDa), respectively, and also for p53 (~53 kDa) and Tfam (~24 kDa). Representative blots of b mitochondrial p53 content (~53 kDa) in the muscle and heart of WT, PolG-SED, PolG-END ( n = 6–8/group), c p53 co-immunoprecipitation ( IP ) followed by immunoblotting ( IB ) for mitochondrial transcription factor A (Tfam; ~24 kDa) to assess mitochondrial p53-Tfam complex content in muscle and heart mitochondria from WT, PolG-SED, PolG-END ( n = 4–5/group), and d p53 co- IP followed by IB for POLG1 (~140 kDa) to assess mitochondrial p53-POLG1 complex content in muscle and heart mitochondria from WT, PolG-SED, and PolG-END ( n = 6–8/group). VDAC (~32 kDa) was used as a mitochondrial loading control. e p53-POLG1-Tfam complex is bound to mtDNA (quantified using two independent mtDNA regions: COX-II and cytochrome b ) in muscle mitochondrial fractions of WT, PolG-SED, and PolG-END mice ( n = 4–6/group). A non-specific IgG antibody was used as negative control antibody. Asterisk (PolG-SED vs. both WT and PolG-END): * P
Figure Legend Snippet: Endurance exercise increases the abundance of p53 in mitochondrial matrix where it interacts with mtDNA in a complex with POLG1 and Tfam in mtDNA mutator mice. a Mitochondrial p53 is primarily localized in the matrix. Muscle mitochondria were subfractionated into outer mitochondrial membrane (OMM), intermembrane space (IMS), inner mitochondrial membrane (IMM), and matrix (Mx) fractions, and these fractions were immunoblotted for the compartment-specific proteins TOMM22 (~16 kDa), cytochrome c (~14 kDa), COX-IV (~17 kDa), and CS (~45 kDa), respectively, and also for p53 (~53 kDa) and Tfam (~24 kDa). Representative blots of b mitochondrial p53 content (~53 kDa) in the muscle and heart of WT, PolG-SED, PolG-END ( n = 6–8/group), c p53 co-immunoprecipitation ( IP ) followed by immunoblotting ( IB ) for mitochondrial transcription factor A (Tfam; ~24 kDa) to assess mitochondrial p53-Tfam complex content in muscle and heart mitochondria from WT, PolG-SED, PolG-END ( n = 4–5/group), and d p53 co- IP followed by IB for POLG1 (~140 kDa) to assess mitochondrial p53-POLG1 complex content in muscle and heart mitochondria from WT, PolG-SED, and PolG-END ( n = 6–8/group). VDAC (~32 kDa) was used as a mitochondrial loading control. e p53-POLG1-Tfam complex is bound to mtDNA (quantified using two independent mtDNA regions: COX-II and cytochrome b ) in muscle mitochondrial fractions of WT, PolG-SED, and PolG-END mice ( n = 4–6/group). A non-specific IgG antibody was used as negative control antibody. Asterisk (PolG-SED vs. both WT and PolG-END): * P

Techniques Used: Mouse Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Negative Control

Endurance exercise reduces random mtDNA somatic mutations, attenuates mitochondrial ROS-mediated oxidative damage, mitigates telomere shortening, and reduces nuclear accumulation of p53 in mtDNA mutator mice. a Random mtDNA somatic mutation rate (per 1000 nucleotides of mtDNA) in muscle ( quadriceps femoris ) WT, PolG-SED, and PolG-END mice ( n = 4–5/group). b H 2 O 2 production rate in muscle mitochondrial fractions of WT, PolG-SED, and PolG-END ( n = 5–7/group). Complex I and II substrates: P/M, pyruvate/malate and SUC, succinate (5 mM each), respectively. Complex I and III inhibitors: ROT, rotenone, and AA, antimycin A (0.5 μM each), respectively. c Protein carbonyls ( PC ) content in muscle ( tibialis anterior ) and heart mitochondrial fractions of WT, PolG-SED, and PolG-END ( n = 5–7/group). d SOD2 and catalase enzyme activity in the muscle ( quadriceps femoris ) and heart of WT, PolG-SED, and PolG-END ( n = 7/group). e Average telomere length ratios in the heart, hematopoietic stem and progenitor cells ( HSC ), and satellite cells ( SC ) of WT, PolG-SED, and PolG-END ( n = 6–8/group). f Representative blots of nuclear p53 content (~53 kDa) in the muscle ( quadriceps femoris ) and heart of WT, PolG-SED, and PolG-END ( n = 5–8/group). Histone H2B (~14 kDa) was used as a nuclear loading control. (PolG-SED vs. both WT and PolG-END) = * P
Figure Legend Snippet: Endurance exercise reduces random mtDNA somatic mutations, attenuates mitochondrial ROS-mediated oxidative damage, mitigates telomere shortening, and reduces nuclear accumulation of p53 in mtDNA mutator mice. a Random mtDNA somatic mutation rate (per 1000 nucleotides of mtDNA) in muscle ( quadriceps femoris ) WT, PolG-SED, and PolG-END mice ( n = 4–5/group). b H 2 O 2 production rate in muscle mitochondrial fractions of WT, PolG-SED, and PolG-END ( n = 5–7/group). Complex I and II substrates: P/M, pyruvate/malate and SUC, succinate (5 mM each), respectively. Complex I and III inhibitors: ROT, rotenone, and AA, antimycin A (0.5 μM each), respectively. c Protein carbonyls ( PC ) content in muscle ( tibialis anterior ) and heart mitochondrial fractions of WT, PolG-SED, and PolG-END ( n = 5–7/group). d SOD2 and catalase enzyme activity in the muscle ( quadriceps femoris ) and heart of WT, PolG-SED, and PolG-END ( n = 7/group). e Average telomere length ratios in the heart, hematopoietic stem and progenitor cells ( HSC ), and satellite cells ( SC ) of WT, PolG-SED, and PolG-END ( n = 6–8/group). f Representative blots of nuclear p53 content (~53 kDa) in the muscle ( quadriceps femoris ) and heart of WT, PolG-SED, and PolG-END ( n = 5–8/group). Histone H2B (~14 kDa) was used as a nuclear loading control. (PolG-SED vs. both WT and PolG-END) = * P

Techniques Used: Mouse Assay, Mutagenesis, Activity Assay

35) Product Images from "Emodin via colonic irrigation modulates gut microbiota and reduces uremic toxins in rats with chronic kidney disease"

Article Title: Emodin via colonic irrigation modulates gut microbiota and reduces uremic toxins in rats with chronic kidney disease

Journal: Oncotarget

doi: 10.18632/oncotarget.8160

Biplot of redundancy analysis (RDA) of the gut microbiota compositions after ECI treatments in CKD rats The uremic toxins of Urea and IS were used as environmental variables. On Top-left, P -value was obtained by Monte Carlo permutation procedure (MCPP).
Figure Legend Snippet: Biplot of redundancy analysis (RDA) of the gut microbiota compositions after ECI treatments in CKD rats The uremic toxins of Urea and IS were used as environmental variables. On Top-left, P -value was obtained by Monte Carlo permutation procedure (MCPP).

Techniques Used:

Quantifications of bacteria in the fecal microbiota (log 10 copies/g stool) Real-time qPCR was performed to detect the fecal microbiota 4 weeks after CCI or ECI treatments following the 5/6 nephrectomy. Data are presented as Mean ± SD, * p
Figure Legend Snippet: Quantifications of bacteria in the fecal microbiota (log 10 copies/g stool) Real-time qPCR was performed to detect the fecal microbiota 4 weeks after CCI or ECI treatments following the 5/6 nephrectomy. Data are presented as Mean ± SD, * p

Techniques Used: Real-time Polymerase Chain Reaction

Weighted Unifrac PCoA analysis of gut microbiota based on the OTU data from pyrosequencing run A point represents a sample from each group. The sample numbers (n) in each group: CTL = 13, CCI = 9, and ECI = 9.
Figure Legend Snippet: Weighted Unifrac PCoA analysis of gut microbiota based on the OTU data from pyrosequencing run A point represents a sample from each group. The sample numbers (n) in each group: CTL = 13, CCI = 9, and ECI = 9.

Techniques Used: CTL Assay

Relative abundance of the gut microbiota A. abundance and prevalence of the different bacterial phyla in each group. B. abundance and prevalence of the different bacterial genera in each group.
Figure Legend Snippet: Relative abundance of the gut microbiota A. abundance and prevalence of the different bacterial phyla in each group. B. abundance and prevalence of the different bacterial genera in each group.

Techniques Used:

36) Product Images from "Ultrasound-Sensitive Liposomes for Triggered Macromolecular Drug Delivery: Formulation and In Vitro Characterization"

Article Title: Ultrasound-Sensitive Liposomes for Triggered Macromolecular Drug Delivery: Formulation and In Vitro Characterization

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2019.01463

Storage stability of USL at 4°C in HBS buffer. Panel (A) shows size measurements, obtained by DLS over a period of 30 days, of nanoparticles (NUSL and USL) loaded with HRP and ML1. Panel (B) shows HRP retention. In both graphs, the colors of the lines and the symbols correspond to the same lipid composition: the red line corresponds to liposomal formulations composed of initial 20 mol% DSPE-PEG 2000 and containing (full line and circle, USL20) or not (dashed line and empty circle, NUSL20) PFC nanoemulsion; the green line corresponds to liposomal formulations composed of initial 10 mol% DSPE-PEG 2000 and containing (full line and square, USL10) or not (dashed line and empty square, NUSL10) PFC nanoemulsion; the blue line corresponds to liposomal formulations composed of initial 5 mol% DSPE-PEG 2000 and containing (full line and diamond, USL5) or not (dashed line and empty diamond, NUSL5) PFC nanoemulsion. Data are the average ± standard deviation of three independent samples.
Figure Legend Snippet: Storage stability of USL at 4°C in HBS buffer. Panel (A) shows size measurements, obtained by DLS over a period of 30 days, of nanoparticles (NUSL and USL) loaded with HRP and ML1. Panel (B) shows HRP retention. In both graphs, the colors of the lines and the symbols correspond to the same lipid composition: the red line corresponds to liposomal formulations composed of initial 20 mol% DSPE-PEG 2000 and containing (full line and circle, USL20) or not (dashed line and empty circle, NUSL20) PFC nanoemulsion; the green line corresponds to liposomal formulations composed of initial 10 mol% DSPE-PEG 2000 and containing (full line and square, USL10) or not (dashed line and empty square, NUSL10) PFC nanoemulsion; the blue line corresponds to liposomal formulations composed of initial 5 mol% DSPE-PEG 2000 and containing (full line and diamond, USL5) or not (dashed line and empty diamond, NUSL5) PFC nanoemulsion. Data are the average ± standard deviation of three independent samples.

Techniques Used: Standard Deviation

37) Product Images from "Development of a Hexaplex PCR Assay for Rapid Detection of Virulence and Regulatory Genes in Vibrio cholerae and Vibrio mimicus"

Article Title: Development of a Hexaplex PCR Assay for Rapid Detection of Virulence and Regulatory Genes in Vibrio cholerae and Vibrio mimicus

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.40.11.4321-4324.2002

(A) Ethidium bromide-stained agarose gel electrophoresis of hexaplex PCR products from Vibrio cholerae strains. Lane M, 100-bp DNA ladder (NEB); lanes 1, 8 and 9, cholera toxin-producing V. cholerae O1 El Tor strain 20, classical strain 569B, and V. cholerae O139 strain ATCC 51394, respectively; lanes 2 through 4, non-cholera toxin-producing V. cholerae O1 El Tor strains X392, 274-80, and 1074-78, respectively; lane 5, tcpA -negative V. cholerae O1 classical strain O395 RT 110-12; lane 6, toxR -negative V. cholerae O1 classical strain O395-12; lane 7, ctxA -negative V. cholerae O1 classical strain CVD 103-HgR. (B) Hexaplex PCR products of representative V. cholerae O1, O139, non-O1, and non-O139 and V. mimicus strains in an ethidium bromide-stained agarose gel. Lane M, 100-bp DNA ladder (NEB); lane 1, cholera toxin-producing V. cholerae O1 El Tor strain KO63 (diarrhea isolate from Kerala, India, 2000); lane 2, non-cholera toxin-producing V. cholerae O1 El Tor strain GS2 (isolate from Gerris spinolae , Varanasi, India, 1987); lane 3, cholera toxin-producing V. cholerae O139 strain VO522 (diarrhea isolate from Varanasi, India, 1994); lane 4, non-cholera toxin-producing V. cholerae O139 strain CO788 (diarrhea isolate from Kolkata, India, 1992); lanes 5 and 6, non-cholera toxin-producing V. cholerae non-O1, non-O139 strains 12475 and 13094, respectively (diarrhea isolates from Varanasi, India, 1979); lanes 7 and 8, non-cholera toxin-producing V. mimicus strains WM18 (water isolate from the River Ganges, Varanasi, India, 1988) and VM2 (water isolate from the River Ganges, Varanasi, India, 1988), respectively; and lane 9, cholera toxin-producing V. mimicus strain WM8 (water isolate from the River Ganges, Varanasi, India, 1986).
Figure Legend Snippet: (A) Ethidium bromide-stained agarose gel electrophoresis of hexaplex PCR products from Vibrio cholerae strains. Lane M, 100-bp DNA ladder (NEB); lanes 1, 8 and 9, cholera toxin-producing V. cholerae O1 El Tor strain 20, classical strain 569B, and V. cholerae O139 strain ATCC 51394, respectively; lanes 2 through 4, non-cholera toxin-producing V. cholerae O1 El Tor strains X392, 274-80, and 1074-78, respectively; lane 5, tcpA -negative V. cholerae O1 classical strain O395 RT 110-12; lane 6, toxR -negative V. cholerae O1 classical strain O395-12; lane 7, ctxA -negative V. cholerae O1 classical strain CVD 103-HgR. (B) Hexaplex PCR products of representative V. cholerae O1, O139, non-O1, and non-O139 and V. mimicus strains in an ethidium bromide-stained agarose gel. Lane M, 100-bp DNA ladder (NEB); lane 1, cholera toxin-producing V. cholerae O1 El Tor strain KO63 (diarrhea isolate from Kerala, India, 2000); lane 2, non-cholera toxin-producing V. cholerae O1 El Tor strain GS2 (isolate from Gerris spinolae , Varanasi, India, 1987); lane 3, cholera toxin-producing V. cholerae O139 strain VO522 (diarrhea isolate from Varanasi, India, 1994); lane 4, non-cholera toxin-producing V. cholerae O139 strain CO788 (diarrhea isolate from Kolkata, India, 1992); lanes 5 and 6, non-cholera toxin-producing V. cholerae non-O1, non-O139 strains 12475 and 13094, respectively (diarrhea isolates from Varanasi, India, 1979); lanes 7 and 8, non-cholera toxin-producing V. mimicus strains WM18 (water isolate from the River Ganges, Varanasi, India, 1988) and VM2 (water isolate from the River Ganges, Varanasi, India, 1988), respectively; and lane 9, cholera toxin-producing V. mimicus strain WM8 (water isolate from the River Ganges, Varanasi, India, 1986).

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

38) Product Images from "Ultrasound-Sensitive Liposomes for Triggered Macromolecular Drug Delivery: Formulation and In Vitro Characterization"

Article Title: Ultrasound-Sensitive Liposomes for Triggered Macromolecular Drug Delivery: Formulation and In Vitro Characterization

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2019.01463

Storage stability of USL at 4°C in HBS buffer. Panel (A) shows size measurements, obtained by DLS over a period of 30 days, of nanoparticles (NUSL and USL) loaded with HRP and ML1. Panel (B) shows HRP retention. In both graphs, the colors of the lines and the symbols correspond to the same lipid composition: the red line corresponds to liposomal formulations composed of initial 20 mol% DSPE-PEG 2000 and containing (full line and circle, USL20) or not (dashed line and empty circle, NUSL20) PFC nanoemulsion; the green line corresponds to liposomal formulations composed of initial 10 mol% DSPE-PEG 2000 and containing (full line and square, USL10) or not (dashed line and empty square, NUSL10) PFC nanoemulsion; the blue line corresponds to liposomal formulations composed of initial 5 mol% DSPE-PEG 2000 and containing (full line and diamond, USL5) or not (dashed line and empty diamond, NUSL5) PFC nanoemulsion. Data are the average ± standard deviation of three independent samples.
Figure Legend Snippet: Storage stability of USL at 4°C in HBS buffer. Panel (A) shows size measurements, obtained by DLS over a period of 30 days, of nanoparticles (NUSL and USL) loaded with HRP and ML1. Panel (B) shows HRP retention. In both graphs, the colors of the lines and the symbols correspond to the same lipid composition: the red line corresponds to liposomal formulations composed of initial 20 mol% DSPE-PEG 2000 and containing (full line and circle, USL20) or not (dashed line and empty circle, NUSL20) PFC nanoemulsion; the green line corresponds to liposomal formulations composed of initial 10 mol% DSPE-PEG 2000 and containing (full line and square, USL10) or not (dashed line and empty square, NUSL10) PFC nanoemulsion; the blue line corresponds to liposomal formulations composed of initial 5 mol% DSPE-PEG 2000 and containing (full line and diamond, USL5) or not (dashed line and empty diamond, NUSL5) PFC nanoemulsion. Data are the average ± standard deviation of three independent samples.

Techniques Used: Standard Deviation

HRP and ML1 release from USL10 and NUSL10 as function of exposure time at fixed negative pressure (24 MPa), n = 4. In both graphs, the full line and full squares correspond to USL10, and the dashed line and empty squares correspond to NUSL10. For each exposure time the difference in HRP (A) or ML1 (B) release between USL10 and NUSL10 samples was significant (multiple t-test for all time points with Holm-Sidak correction).
Figure Legend Snippet: HRP and ML1 release from USL10 and NUSL10 as function of exposure time at fixed negative pressure (24 MPa), n = 4. In both graphs, the full line and full squares correspond to USL10, and the dashed line and empty squares correspond to NUSL10. For each exposure time the difference in HRP (A) or ML1 (B) release between USL10 and NUSL10 samples was significant (multiple t-test for all time points with Holm-Sidak correction).

Techniques Used:

HRP release profiles from (A) NUSL (squares), (B) USL (circles) and (C) NUSL (stars) spiked with nanoemulsion, and ML1 release profiles from (D) NUSL formulations (squares) and (E) USL. All samples were exposure to HIFU for 1 min and variable negative pressure (2–24 MPa) and subsequently analyzed. In all graphs, the colors of the lines correspond to the same lipid composition: the red line corresponds to liposomal formulations composed of initial 20% mol DSPE-PEG2000; the green line corresponds to liposomal formulations composed of initial 10% mol DSPE-PEG2000; the blue line corresponds to liposomal formulations composed of initial 5% mol DSPE-PEG2000. Background release (i.e., without HIFU) was insignificant (
Figure Legend Snippet: HRP release profiles from (A) NUSL (squares), (B) USL (circles) and (C) NUSL (stars) spiked with nanoemulsion, and ML1 release profiles from (D) NUSL formulations (squares) and (E) USL. All samples were exposure to HIFU for 1 min and variable negative pressure (2–24 MPa) and subsequently analyzed. In all graphs, the colors of the lines correspond to the same lipid composition: the red line corresponds to liposomal formulations composed of initial 20% mol DSPE-PEG2000; the green line corresponds to liposomal formulations composed of initial 10% mol DSPE-PEG2000; the blue line corresponds to liposomal formulations composed of initial 5% mol DSPE-PEG2000. Background release (i.e., without HIFU) was insignificant (

Techniques Used:

39) Product Images from "Identification, Classification and Differential Expression of Oleosin Genes in Tung Tree (Vernicia fordii)"

Article Title: Identification, Classification and Differential Expression of Oleosin Genes in Tung Tree (Vernicia fordii)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0088409

qPCR optimization, specificity and efficiency for OLE assay. (A) TaqMan qPCR optimization. TaqMan qPCR reactions contained 5 ng RNA-equivalent cDNA from tung seeds, various concentrations of the primers and TaqMan probe. Ole1 assay optimization is presented. (B) Specificity of SYBR Green qPCR by melt curve analysis and gel electrophoresis of amplification products. The qPCR reactions contained 5 ng RNA-equivalent cDNA from tung tree seeds. The qPCR products were separated by agarose gel electrophoresis. Lane 100 bp represents DNA ladders with 100 bp as the smallest band, increasing upward in 100 bp increments. The results using RNA isolated from leaves and flowers are presented in Figure S3 . (C) qPCR efficiency for OLE assay. TaqMan and SYBR Green qPCR reaction mixtures contained variable concentrations of RNA-equivalent cDNA from tung seeds, the optimized concentrations of each primer and probe (200 nM), and Absolute QPCR Mix (TaqMan qPCR) or each primer and 1 x iQ SYBR Green Supermix (SYBR Green qPCR). The results using RNA isolated from stage 4 seeds of tree 1 are shown in the figure. The results for Ole2, Ole3 and Ole4 assays are presented in Figure S4 . The results using RNA from other stages of tung seeds, leaves and flowers are presented in Table S1 .
Figure Legend Snippet: qPCR optimization, specificity and efficiency for OLE assay. (A) TaqMan qPCR optimization. TaqMan qPCR reactions contained 5 ng RNA-equivalent cDNA from tung seeds, various concentrations of the primers and TaqMan probe. Ole1 assay optimization is presented. (B) Specificity of SYBR Green qPCR by melt curve analysis and gel electrophoresis of amplification products. The qPCR reactions contained 5 ng RNA-equivalent cDNA from tung tree seeds. The qPCR products were separated by agarose gel electrophoresis. Lane 100 bp represents DNA ladders with 100 bp as the smallest band, increasing upward in 100 bp increments. The results using RNA isolated from leaves and flowers are presented in Figure S3 . (C) qPCR efficiency for OLE assay. TaqMan and SYBR Green qPCR reaction mixtures contained variable concentrations of RNA-equivalent cDNA from tung seeds, the optimized concentrations of each primer and probe (200 nM), and Absolute QPCR Mix (TaqMan qPCR) or each primer and 1 x iQ SYBR Green Supermix (SYBR Green qPCR). The results using RNA isolated from stage 4 seeds of tree 1 are shown in the figure. The results for Ole2, Ole3 and Ole4 assays are presented in Figure S4 . The results using RNA from other stages of tung seeds, leaves and flowers are presented in Table S1 .

Techniques Used: Real-time Polymerase Chain Reaction, SYBR Green Assay, Nucleic Acid Electrophoresis, Amplification, Agarose Gel Electrophoresis, Isolation

40) Product Images from "The Use of Nanotrap Particles Technology in Capturing HIV-1 Virions and Viral Proteins from Infected Cells"

Article Title: The Use of Nanotrap Particles Technology in Capturing HIV-1 Virions and Viral Proteins from Infected Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0096778

HIV-1 and exosomes capturing capacity of nanoparticles. ( A ) 100 µl aliquot of the dual-tropic HIV-1 89.6 containing 10 or 1 ng p24/ml was incubated with a pellet of 0.02, 0.2 or 2.0 mg of NT086 for 30 min at room temperature. The virus bound-nanoparticles were washed and RNA isolated. The RNA was converted to cDNA and PCR reaction mixtures were prepared using high-low concentrations of cDNA, Gag primers and the iTaq Universal SYBR Green Supermix. Serial dilutions of DNA from 8E5 cells were used as the standards. Quantitative real-time PCR reactions were carried out in triplicate. The asterix represents the statistical significance at the level of p
Figure Legend Snippet: HIV-1 and exosomes capturing capacity of nanoparticles. ( A ) 100 µl aliquot of the dual-tropic HIV-1 89.6 containing 10 or 1 ng p24/ml was incubated with a pellet of 0.02, 0.2 or 2.0 mg of NT086 for 30 min at room temperature. The virus bound-nanoparticles were washed and RNA isolated. The RNA was converted to cDNA and PCR reaction mixtures were prepared using high-low concentrations of cDNA, Gag primers and the iTaq Universal SYBR Green Supermix. Serial dilutions of DNA from 8E5 cells were used as the standards. Quantitative real-time PCR reactions were carried out in triplicate. The asterix represents the statistical significance at the level of p

Techniques Used: Incubation, Isolation, Polymerase Chain Reaction, SYBR Green Assay, Real-time Polymerase Chain Reaction

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    Expression of milk fat globule-EGF-factor 8 (MFG-E8) in placental development during pregnancy. ( a – d ) Hematoxylin and eosin (H E) staining and immunofluorescent labeling of the developing placenta with anti-CD34 and anti-MFG-E8 antibodies at embryonic day (E)11.5. High magnification images of the boxed areas are shown in the upper right corner of each panels. Scale bar: 50 μm. ( e ) real-time reverse transcription-polymerase chain reaction <t>(RT-PCR)</t> analysis of MFG-E8 mRNA in the placenta at different days of gestation. ( f ) quantitative PCR (qPCR) analysis of MFG-E8 mRNA in the placenta at different days of gestation. The expression level was presented as relative expressions (fold changes) over the lowest value (the lowest level was assigned a value of 1) after normalization to β-actin expression.
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    Expression of milk fat globule-EGF-factor 8 (MFG-E8) in placental development during pregnancy. ( a – d ) Hematoxylin and eosin (H E) staining and immunofluorescent labeling of the developing placenta with anti-CD34 and anti-MFG-E8 antibodies at embryonic day (E)11.5. High magnification images of the boxed areas are shown in the upper right corner of each panels. Scale bar: 50 μm. ( e ) real-time reverse transcription-polymerase chain reaction <t>(RT-PCR)</t> analysis of MFG-E8 mRNA in the placenta at different days of gestation. ( f ) quantitative PCR (qPCR) analysis of MFG-E8 mRNA in the placenta at different days of gestation. The expression level was presented as relative expressions (fold changes) over the lowest value (the lowest level was assigned a value of 1) after normalization to β-actin expression.
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    Expression of milk fat globule-EGF-factor 8 (MFG-E8) in placental development during pregnancy. ( a – d ) Hematoxylin and eosin (H E) staining and immunofluorescent labeling of the developing placenta with anti-CD34 and anti-MFG-E8 antibodies at embryonic day (E)11.5. High magnification images of the boxed areas are shown in the upper right corner of each panels. Scale bar: 50 μm. ( e ) real-time reverse transcription-polymerase chain reaction <t>(RT-PCR)</t> analysis of MFG-E8 mRNA in the placenta at different days of gestation. ( f ) quantitative PCR (qPCR) analysis of MFG-E8 mRNA in the placenta at different days of gestation. The expression level was presented as relative expressions (fold changes) over the lowest value (the lowest level was assigned a value of 1) after normalization to β-actin expression.
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    Expression of milk fat globule-EGF-factor 8 (MFG-E8) in placental development during pregnancy. ( a – d ) Hematoxylin and eosin (H E) staining and immunofluorescent labeling of the developing placenta with anti-CD34 and anti-MFG-E8 antibodies at embryonic day (E)11.5. High magnification images of the boxed areas are shown in the upper right corner of each panels. Scale bar: 50 μm. ( e ) real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis of MFG-E8 mRNA in the placenta at different days of gestation. ( f ) quantitative PCR (qPCR) analysis of MFG-E8 mRNA in the placenta at different days of gestation. The expression level was presented as relative expressions (fold changes) over the lowest value (the lowest level was assigned a value of 1) after normalization to β-actin expression.

    Journal: Experimental & Molecular Medicine

    Article Title: Fetal hematopoietic stem cells express MFG-E8 during mouse embryogenesis

    doi: 10.1038/emm.2015.42

    Figure Lengend Snippet: Expression of milk fat globule-EGF-factor 8 (MFG-E8) in placental development during pregnancy. ( a – d ) Hematoxylin and eosin (H E) staining and immunofluorescent labeling of the developing placenta with anti-CD34 and anti-MFG-E8 antibodies at embryonic day (E)11.5. High magnification images of the boxed areas are shown in the upper right corner of each panels. Scale bar: 50 μm. ( e ) real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis of MFG-E8 mRNA in the placenta at different days of gestation. ( f ) quantitative PCR (qPCR) analysis of MFG-E8 mRNA in the placenta at different days of gestation. The expression level was presented as relative expressions (fold changes) over the lowest value (the lowest level was assigned a value of 1) after normalization to β-actin expression.

    Article Snippet: For real-time quantitative PCR (qPCR) analysis, real-time reverse transcription-polymerase chain reaction (RT-PCR) mixtures were performed in duplicate with the iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA).

    Techniques: Expressing, Staining, Labeling, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Expression of milk fat globule-EGF-factor 8 (MFG-E8) in CD34 + cells in the fetal liver. ( a and b ) Flow cytometric analysis of CD34 + cells in the developing liver before ( a ) and after ( b ) purification with anti-CD34 antibody. ( c ) quantitative PCR (qPCR) analysis of sorted cells for the expression of Cd34 . Cd34 expression in the CD34 + fraction was presented as relative expressions (fold changes) over CD34 − fraction after normalization to β-actin expression. * P

    Journal: Experimental & Molecular Medicine

    Article Title: Fetal hematopoietic stem cells express MFG-E8 during mouse embryogenesis

    doi: 10.1038/emm.2015.42

    Figure Lengend Snippet: Expression of milk fat globule-EGF-factor 8 (MFG-E8) in CD34 + cells in the fetal liver. ( a and b ) Flow cytometric analysis of CD34 + cells in the developing liver before ( a ) and after ( b ) purification with anti-CD34 antibody. ( c ) quantitative PCR (qPCR) analysis of sorted cells for the expression of Cd34 . Cd34 expression in the CD34 + fraction was presented as relative expressions (fold changes) over CD34 − fraction after normalization to β-actin expression. * P

    Article Snippet: For real-time quantitative PCR (qPCR) analysis, real-time reverse transcription-polymerase chain reaction (RT-PCR) mixtures were performed in duplicate with the iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA).

    Techniques: Expressing, Flow Cytometry, Purification, Real-time Polymerase Chain Reaction