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    Structured Review

    Thermo Fisher pcr amplification
    Results of methylation‐specific <t>PCR</t> ( MS ‐ PCR ) for GSTP 1 promoter region. Lanes U and M correspond to amplification with primers recognizing unmethylated (97 bp) and methylated (91 bp) sequences, respectively. <t>DNA</t> from MCF ‐7 cell line was used as positive control for methylation and DNA from BCPAP cell line was used as negative control. Results of normal bladder urotheliums (C1, C2: Control 1, 2) and the 30 BC cases (1–30) are represented. Water was used as negative PCR control (H 2 O). On the right side: the 100 bp DNA ladder. GSTP1 : NM_000852.3 . BC, bladder cancer
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    Images

    1) Product Images from "Evaluation of glutathione S‐transferase pi 1 expression and gene promoter methylation in Moroccan patients with urothelial bladder cancer, et al. Evaluation of glutathione S‐transferase pi 1 expression and gene promoter methylation in Moroccan patients with urothelial bladder cancer"

    Article Title: Evaluation of glutathione S‐transferase pi 1 expression and gene promoter methylation in Moroccan patients with urothelial bladder cancer, et al. Evaluation of glutathione S‐transferase pi 1 expression and gene promoter methylation in Moroccan patients with urothelial bladder cancer

    Journal: Molecular Genetics & Genomic Medicine

    doi: 10.1002/mgg3.449

    Results of methylation‐specific PCR ( MS ‐ PCR ) for GSTP 1 promoter region. Lanes U and M correspond to amplification with primers recognizing unmethylated (97 bp) and methylated (91 bp) sequences, respectively. DNA from MCF ‐7 cell line was used as positive control for methylation and DNA from BCPAP cell line was used as negative control. Results of normal bladder urotheliums (C1, C2: Control 1, 2) and the 30 BC cases (1–30) are represented. Water was used as negative PCR control (H 2 O). On the right side: the 100 bp DNA ladder. GSTP1 : NM_000852.3 . BC, bladder cancer
    Figure Legend Snippet: Results of methylation‐specific PCR ( MS ‐ PCR ) for GSTP 1 promoter region. Lanes U and M correspond to amplification with primers recognizing unmethylated (97 bp) and methylated (91 bp) sequences, respectively. DNA from MCF ‐7 cell line was used as positive control for methylation and DNA from BCPAP cell line was used as negative control. Results of normal bladder urotheliums (C1, C2: Control 1, 2) and the 30 BC cases (1–30) are represented. Water was used as negative PCR control (H 2 O). On the right side: the 100 bp DNA ladder. GSTP1 : NM_000852.3 . BC, bladder cancer

    Techniques Used: Methylation, Polymerase Chain Reaction, Mass Spectrometry, Amplification, Positive Control, Negative Control

    2) Product Images from "Potent suppression of HIV-1 cell attachment by Kudzu root extract"

    Article Title: Potent suppression of HIV-1 cell attachment by Kudzu root extract

    Journal: Retrovirology

    doi: 10.1186/s12977-018-0446-x

    Kudzu inhibits HIV-1 replication of X4 tropic viruses. a , b Activity of Kudzu in acute infection of HeLa-CD4-LTR-LacZ cells with NL4-3. HeLa-CD4-LTR-LacZ cells were infected with HIV-1 NL4-3 strain in the presence of different dilutions of Kudzu. a β-Gal activity was measured 72 h later. Untreat.: untreated. The mean ± SD of 5 independent experiments is represented. b Viral supernatants recovered 72 h post infection were assayed for their p24 antigen content using a sandwich ELISA kit. Untreat.: untreated. Data is a mean ± SD of 2 independent experiments. c Impact of Kudzu on HIV-1 integration. HeLa-CD4-LTR-LacZ were infected with NL4-3 in presence of compounds for 24 h. Next day, DNA was extracted and provirus integration was quantified by Alu-PCR followed by qPCR. Saquinavir (Saq., a protease inhibitor, 200 nM), Efavirenz (Efav., a reverse transcriptase inhibitor, 200 nM), Raltegravir (Ralt., an integrase inhibitor, 200 nM), and AMD3100 (an entry inhibitor, 10 nM) were used as controls. Kudzu was used at 1:200. The mean ± SD of 5 independent experiments is represented. d Impact of Kudzu on HIV-1 integration in primary CD4 + T cells 24 h post-infection and treatment. A cocktail of antiretrovirals (ARVS: 180 nM AZT, a reverse transcriptase inhibitor, 100 nM Efavirenz and 200 nM Raltegravir) and Enfuvirtide (1 μg/ml) were used as controls. Shown is the mean ± SD of 3 independent experiments. e Activity of Kudzu on reverse-transcription products of HIV-1 10 h post-infection. HeLa-CD4-LTR-LacZ were infected with NL4-3 in presence of compounds for 10 h. Next, DNA was extracted and early and late RT products were measured by qPCR. Kudzu was used at 1:200. Error Bars from qPCR (n = 3) ± SD from 2 independent experiments for early products. The mean ± SD of 4 independent experiments is represented for late products. f Time-of-addition experiment of kudzu in HeLa-CD4-LTR-LacZ infected with NL4-3 strain. Kudzu or control compounds (4 nM AMD3100 and 10 nM Efavirenz) were added at 1, 2, 3, 4, 5 and 6 h postinfection. β-Gal activity was measured 72 h later. Data shown is representative of 2 independent experiments. The two-tailed paired t test was used for statistical comparisons. *: p value
    Figure Legend Snippet: Kudzu inhibits HIV-1 replication of X4 tropic viruses. a , b Activity of Kudzu in acute infection of HeLa-CD4-LTR-LacZ cells with NL4-3. HeLa-CD4-LTR-LacZ cells were infected with HIV-1 NL4-3 strain in the presence of different dilutions of Kudzu. a β-Gal activity was measured 72 h later. Untreat.: untreated. The mean ± SD of 5 independent experiments is represented. b Viral supernatants recovered 72 h post infection were assayed for their p24 antigen content using a sandwich ELISA kit. Untreat.: untreated. Data is a mean ± SD of 2 independent experiments. c Impact of Kudzu on HIV-1 integration. HeLa-CD4-LTR-LacZ were infected with NL4-3 in presence of compounds for 24 h. Next day, DNA was extracted and provirus integration was quantified by Alu-PCR followed by qPCR. Saquinavir (Saq., a protease inhibitor, 200 nM), Efavirenz (Efav., a reverse transcriptase inhibitor, 200 nM), Raltegravir (Ralt., an integrase inhibitor, 200 nM), and AMD3100 (an entry inhibitor, 10 nM) were used as controls. Kudzu was used at 1:200. The mean ± SD of 5 independent experiments is represented. d Impact of Kudzu on HIV-1 integration in primary CD4 + T cells 24 h post-infection and treatment. A cocktail of antiretrovirals (ARVS: 180 nM AZT, a reverse transcriptase inhibitor, 100 nM Efavirenz and 200 nM Raltegravir) and Enfuvirtide (1 μg/ml) were used as controls. Shown is the mean ± SD of 3 independent experiments. e Activity of Kudzu on reverse-transcription products of HIV-1 10 h post-infection. HeLa-CD4-LTR-LacZ were infected with NL4-3 in presence of compounds for 10 h. Next, DNA was extracted and early and late RT products were measured by qPCR. Kudzu was used at 1:200. Error Bars from qPCR (n = 3) ± SD from 2 independent experiments for early products. The mean ± SD of 4 independent experiments is represented for late products. f Time-of-addition experiment of kudzu in HeLa-CD4-LTR-LacZ infected with NL4-3 strain. Kudzu or control compounds (4 nM AMD3100 and 10 nM Efavirenz) were added at 1, 2, 3, 4, 5 and 6 h postinfection. β-Gal activity was measured 72 h later. Data shown is representative of 2 independent experiments. The two-tailed paired t test was used for statistical comparisons. *: p value

    Techniques Used: Activity Assay, Infection, Sandwich ELISA, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Protease Inhibitor, Two Tailed Test

    3) Product Images from "The inflammasome component Nlrp3 impairs antitumor vaccine by enhancing the accumulation of tumor-associated myeloid-derived suppressor cells"

    Article Title: The inflammasome component Nlrp3 impairs antitumor vaccine by enhancing the accumulation of tumor-associated myeloid-derived suppressor cells

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-10-1921

    Nlrp3 is expressed by tumor-associated myeloid cells Expression of Nlrp3 by real time PCR in (1) Gr-1 + , CD11b + (2) Gr-1 − , CD11b + and (3) Gr-1 − , CD11b − sorted cells. The panel on left is a representative dot plot of sorted cells from a subcutaneous B16-F10 tumor in a vaccinated WT (A) and Nlrp3 −/− mouse (B). The panel on the right is a bar graph of Nlrp3 transcripts normalized to cell number.
    Figure Legend Snippet: Nlrp3 is expressed by tumor-associated myeloid cells Expression of Nlrp3 by real time PCR in (1) Gr-1 + , CD11b + (2) Gr-1 − , CD11b + and (3) Gr-1 − , CD11b − sorted cells. The panel on left is a representative dot plot of sorted cells from a subcutaneous B16-F10 tumor in a vaccinated WT (A) and Nlrp3 −/− mouse (B). The panel on the right is a bar graph of Nlrp3 transcripts normalized to cell number.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    4) Product Images from "Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism"

    Article Title: Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

    Journal: Journal of Biomedicine and Biotechnology

    doi: 10.1155/2012/593195

    Effect of camel milk on apoptotic and oxidative stress markers Caspase-3 (a), DR4 (b), and HO-1 (c) mRNA levels in MCF7 cells. MCF7 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of Caspase-3, DR4, and HO-1 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P
    Figure Legend Snippet: Effect of camel milk on apoptotic and oxidative stress markers Caspase-3 (a), DR4 (b), and HO-1 (c) mRNA levels in MCF7 cells. MCF7 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of Caspase-3, DR4, and HO-1 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction

    Effect of camel milk on apoptotic markers Caspase-3 (a), p53 (b), BcL2 (c), and DR4 (d) mRNA levels in HepG2 cells. HepG2 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of Caspase-3, p53, BcL2, and DR4 were quantified using RT-PCR normalized to β -actin housekeeping gene as described in Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P
    Figure Legend Snippet: Effect of camel milk on apoptotic markers Caspase-3 (a), p53 (b), BcL2 (c), and DR4 (d) mRNA levels in HepG2 cells. HepG2 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of Caspase-3, p53, BcL2, and DR4 were quantified using RT-PCR normalized to β -actin housekeeping gene as described in Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction

    Effect of MAPKs inhibitors on camel milk mediated induction of caspase-3 mRNA levels in HepG2 cells. HepG2 cells were pre-treated with for 2 h with JNK inhibitor, SP600125, p38 inhibitor, SB203580, and ERK inhibitor, U0126, before the addition of camel milk (10 mg/mL) for additional 6 h. Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of Caspase-3 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P
    Figure Legend Snippet: Effect of MAPKs inhibitors on camel milk mediated induction of caspase-3 mRNA levels in HepG2 cells. HepG2 cells were pre-treated with for 2 h with JNK inhibitor, SP600125, p38 inhibitor, SB203580, and ERK inhibitor, U0126, before the addition of camel milk (10 mg/mL) for additional 6 h. Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of Caspase-3 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction

    Effect of camel milk on oxidative stress markers HO-1 (a) and NQO1 (b) mRNA levels, and ROS (c) production in HepG2 cells. (a) and (b) HepG2 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of HO-1 and NQO1 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P
    Figure Legend Snippet: Effect of camel milk on oxidative stress markers HO-1 (a) and NQO1 (b) mRNA levels, and ROS (c) production in HepG2 cells. (a) and (b) HepG2 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of HO-1 and NQO1 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction

    Effect of RNA synthesis inhibitor Act-D on the induction Caspase-3 activity by camel milk in HepG2 cells. HepG2 cells were treated with 5 μ g/mL Act-D, a RNA synthesis inhibitor, 30 min before exposure to camel milk (20 mg/mL) for additional 6 h. The amount of Caspase-3 mRNA was quantified using RT-PCR and normalized to β -actin housekeeping gene. Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three inv experiments. + P
    Figure Legend Snippet: Effect of RNA synthesis inhibitor Act-D on the induction Caspase-3 activity by camel milk in HepG2 cells. HepG2 cells were treated with 5 μ g/mL Act-D, a RNA synthesis inhibitor, 30 min before exposure to camel milk (20 mg/mL) for additional 6 h. The amount of Caspase-3 mRNA was quantified using RT-PCR and normalized to β -actin housekeeping gene. Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three inv experiments. + P

    Techniques Used: Activated Clotting Time Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction

    5) Product Images from "Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region"

    Article Title: Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki1001

    Restriction-PCR-assay of the IRF-4 promoter in hematopoietic cells. ( A ) Simplified structure of the CpG sites in the human IRF-4 promoter region including exon 1. Each CpG motif is shown as circle and numbered (above, labeling beginning from the 3′ end moving upstream). Restriction sites, fragments (F1, F2) of the restriction-PCR-assay and regions amplified after bisulfite treatment for sequencing are shown below. The numbers in italics refer to the sequence data by Grossman et al . ( 30 ). ( B and C ) Restriction-PCR-assay. Restriction of DNA with EcoRI (E, no recognition site), HpaII (H, sensitive), Bsp143II (BII, sensitive), Bsh1236I (BI, sensitive) and MspI (M, resistant) and subsequent PCR amplification. Three representative IRF-4-negative (K-562, CML-T1, EM-2) and two IRF-4-positive cell lines (SD-1, BV-173) are shown. IRF-4 expression is denoted on the right. ( B ) PCR-fragment 1 (F1); ( C ) PCR-fragment 2 (F2).
    Figure Legend Snippet: Restriction-PCR-assay of the IRF-4 promoter in hematopoietic cells. ( A ) Simplified structure of the CpG sites in the human IRF-4 promoter region including exon 1. Each CpG motif is shown as circle and numbered (above, labeling beginning from the 3′ end moving upstream). Restriction sites, fragments (F1, F2) of the restriction-PCR-assay and regions amplified after bisulfite treatment for sequencing are shown below. The numbers in italics refer to the sequence data by Grossman et al . ( 30 ). ( B and C ) Restriction-PCR-assay. Restriction of DNA with EcoRI (E, no recognition site), HpaII (H, sensitive), Bsp143II (BII, sensitive), Bsh1236I (BI, sensitive) and MspI (M, resistant) and subsequent PCR amplification. Three representative IRF-4-negative (K-562, CML-T1, EM-2) and two IRF-4-positive cell lines (SD-1, BV-173) are shown. IRF-4 expression is denoted on the right. ( B ) PCR-fragment 1 (F1); ( C ) PCR-fragment 2 (F2).

    Techniques Used: Polymerase Chain Reaction, Labeling, Amplification, Sequencing, Expressing

    6) Product Images from "Genome-wide identification and functional analyses of microRNA signatures associated with cancer pain"

    Article Title: Genome-wide identification and functional analyses of microRNA signatures associated with cancer pain

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201302797

    Manipulation of miRNA expression in lumbar DRGs in mice in vivo via intrathecal application of miRNA mimics and inhibitors Experimental scheme established in this study which enables effective knockdown/induction of miRNA expression in DRGs in vivo and analysis of tumour pain-associated behaviours. Microscopic analyses of whole-mount DRGs or cryosections showing uptake of FAM-conjugated miRNA inhibitors. Scale bar is 50 µm in all panels. Typical examples of qRT-PCR verification of efficacy of miRNA inhibitors in reversing tumour-induced upregulation of miRNAs in ipsilateral DRGs in vivo . Typical examples of qRT-PCR verification of efficacy of miRNA mimics in reversing tumour-induced downregulation of miRNAs and inducing overexpression of miRNAs in ipsilateral DRGs in vivo . In panel (C), * denotes p = 0.02 for miR-1a-3p, 0.04 for miR-34c-3p as compared to corresponding mismatch inhibitor and in panel (D), * denotes p = 0.001 for miR-370-3p and
    Figure Legend Snippet: Manipulation of miRNA expression in lumbar DRGs in mice in vivo via intrathecal application of miRNA mimics and inhibitors Experimental scheme established in this study which enables effective knockdown/induction of miRNA expression in DRGs in vivo and analysis of tumour pain-associated behaviours. Microscopic analyses of whole-mount DRGs or cryosections showing uptake of FAM-conjugated miRNA inhibitors. Scale bar is 50 µm in all panels. Typical examples of qRT-PCR verification of efficacy of miRNA inhibitors in reversing tumour-induced upregulation of miRNAs in ipsilateral DRGs in vivo . Typical examples of qRT-PCR verification of efficacy of miRNA mimics in reversing tumour-induced downregulation of miRNAs and inducing overexpression of miRNAs in ipsilateral DRGs in vivo . In panel (C), * denotes p = 0.02 for miR-1a-3p, 0.04 for miR-34c-3p as compared to corresponding mismatch inhibitor and in panel (D), * denotes p = 0.001 for miR-370-3p and

    Techniques Used: Expressing, Mouse Assay, In Vivo, Quantitative RT-PCR, Over Expression

    7) Product Images from "An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants"

    Article Title: An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-5-30

    Schematic illustration of mutant fragment generation by overlap extension PCR . During first round PCR (PCR1), the 5' and 3' ends of the target genes, as well as the gentamycin (Gm) resistance cassette are amplified using four gene-specific primers (G-UpF-GWL, G-UpR-Gm, G-DnF-Gm and G-DnR-GWR) and the common Gm-specific primers (Gm-F and Gm-R). This generates three fragments with partial overlaps either to each other (indicated by the blue boxes signifying Gm overlap) or the attB1 and attB2 λ recombination sites (indicated by the green and pink boxes). These purified fragments are then assembled in vitro by overlap extension during second round PCR (PCR2) using common primers GW- attB 1 and GW- attB 2, resulting in a recombination-proficient mutant PCR fragment.
    Figure Legend Snippet: Schematic illustration of mutant fragment generation by overlap extension PCR . During first round PCR (PCR1), the 5' and 3' ends of the target genes, as well as the gentamycin (Gm) resistance cassette are amplified using four gene-specific primers (G-UpF-GWL, G-UpR-Gm, G-DnF-Gm and G-DnR-GWR) and the common Gm-specific primers (Gm-F and Gm-R). This generates three fragments with partial overlaps either to each other (indicated by the blue boxes signifying Gm overlap) or the attB1 and attB2 λ recombination sites (indicated by the green and pink boxes). These purified fragments are then assembled in vitro by overlap extension during second round PCR (PCR2) using common primers GW- attB 1 and GW- attB 2, resulting in a recombination-proficient mutant PCR fragment.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Purification, In Vitro

    Gateway-recombinational cloning and return of the plasmid-borne deletion allele to the P. aeruginosa chromosome . The mutant DNA fragment generated by overlap extension PCR is first cloned into pDONR221 via the BP clonase reaction to create the entry clone pDONR221- Gene ::Gm, which then serves as the substrate for LR clonase-mediated recombination into the destination vector pEX18ApGW. The resulting suicide vector pEX18ApGW- Gene ::Gm is then transferred to P. aeruginosa and the plasmid-borne deletion mutation is exchanged with the chromosome to generate the desired deletion mutant. Please note that, as discussed in the text, gene replacement by double-crossover can occur quite frequently, but it can also be a rare event in which case allele exchange happens in two steps involving homologous recombination. First, the suicide plasmid is integrated via a single-crossover event resulting in generation of a merodiploid containing the wild-type and mutant allele. Second, the merodiploid state is resolved by sacB -mediated sucrose counterselection in the presence of gentamycin, resulting in generation of the illustrated chromosomal deletion mutant. An unmarked mutant is then obtained after Flp recombinase-mediated excision of the Gm marker.
    Figure Legend Snippet: Gateway-recombinational cloning and return of the plasmid-borne deletion allele to the P. aeruginosa chromosome . The mutant DNA fragment generated by overlap extension PCR is first cloned into pDONR221 via the BP clonase reaction to create the entry clone pDONR221- Gene ::Gm, which then serves as the substrate for LR clonase-mediated recombination into the destination vector pEX18ApGW. The resulting suicide vector pEX18ApGW- Gene ::Gm is then transferred to P. aeruginosa and the plasmid-borne deletion mutation is exchanged with the chromosome to generate the desired deletion mutant. Please note that, as discussed in the text, gene replacement by double-crossover can occur quite frequently, but it can also be a rare event in which case allele exchange happens in two steps involving homologous recombination. First, the suicide plasmid is integrated via a single-crossover event resulting in generation of a merodiploid containing the wild-type and mutant allele. Second, the merodiploid state is resolved by sacB -mediated sucrose counterselection in the presence of gentamycin, resulting in generation of the illustrated chromosomal deletion mutant. An unmarked mutant is then obtained after Flp recombinase-mediated excision of the Gm marker.

    Techniques Used: Clone Assay, Plasmid Preparation, Mutagenesis, Generated, Polymerase Chain Reaction, Homologous Recombination, Marker

    8) Product Images from "Two RND proteins involved in heavy metal efflux in Caulobacter crescentus belong to separate clusters within proteobacteria"

    Article Title: Two RND proteins involved in heavy metal efflux in Caulobacter crescentus belong to separate clusters within proteobacteria

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-13-79

    Characterization of the czr and ncz promoter regions. ( A ) Beta-galactosidase activity assay of transcription fusions of P czr and P ncz to the lacZ reporter gene. Cells were grown in PYE medium and samples were taken at midlog phase and stationary phase (24 h) for assaying β-galactosidase as described [ 38 ]. The background activity for plasmid alone is around 200 Miller Units. Asterisks indicate results significantly different between the two growth phases within each promoter fusion (p ≤ 0.05). ( B ) Determination of co-transcription of CCNA02805 and CCNA_02806 by amplification with primers RND3 and RND4. Lane 1, PCR amplification using cDNA previously synthesized with Reverse Transcriptase from total RNA from the NA1000 strain; lane 2, PCR amplification from total NA1000 genomic DNA (positive control); lane 3, PCR amplification from total RNA from the NA1000 strain (negative control). The 0.43 kb fragment corresponding to the amplified products is indicated.
    Figure Legend Snippet: Characterization of the czr and ncz promoter regions. ( A ) Beta-galactosidase activity assay of transcription fusions of P czr and P ncz to the lacZ reporter gene. Cells were grown in PYE medium and samples were taken at midlog phase and stationary phase (24 h) for assaying β-galactosidase as described [ 38 ]. The background activity for plasmid alone is around 200 Miller Units. Asterisks indicate results significantly different between the two growth phases within each promoter fusion (p ≤ 0.05). ( B ) Determination of co-transcription of CCNA02805 and CCNA_02806 by amplification with primers RND3 and RND4. Lane 1, PCR amplification using cDNA previously synthesized with Reverse Transcriptase from total RNA from the NA1000 strain; lane 2, PCR amplification from total NA1000 genomic DNA (positive control); lane 3, PCR amplification from total RNA from the NA1000 strain (negative control). The 0.43 kb fragment corresponding to the amplified products is indicated.

    Techniques Used: Activity Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Synthesized, Positive Control, Negative Control

    9) Product Images from "Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium"

    Article Title: Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002804

    Schematic diagram and reproducibility of M-TraM. (A) Schematic overview of the M-TraM screening. In yellow: inverted terminal repeats (ITRs) of the himar1 transposon with outward-facing T7 promoters; in blue: the gentamicin resistance gene in the transposon. Genomic DNA is isolated from the E. faecium mutant library. DNA is digested with the restriction enzyme Alu I, and the DNA fragments are circularized by self-ligation. The transposon-chromosome junction together with an ITR and a T7 promoter is amplified by PCR with primers (blue arrow) that hybridize to the transposon. To eliminate foreign DNA fragments that ligated into the circularized DNA of transposon-chromosome junctions, the PCR products were re-digested with Alu I. The purified DNA fragments are used as template in the in vitro transcription reaction. The resulting RNA products are reverse transcribed into cDNA. After labelling, the cDNA is used for microarray hybridization. (B) Schematic overview of the screening strategy to identify conditionally essential genes by M-TraM. A chromosomal region encompassing three genes (A, B, and C) from three different mutants (1, 2, and 3) is shown. Each mutant carries a single transposon insertion (blue) that disrupts the function of the gene. Mutant libraries are grown in a control condition ( e.g. , BHI) and a test condition ( e.g. , in the presence of ampicillin). All the three genes are non-essential for growth in the control condition. Gene B is required only for the test condition, so mutant 2 exhibits attenuated growth or poorer survival only in the test condition, and will consequently be reduced or be entirely lost from this library (indicated by light shading). M-TraM samples are generated from the two conditions, labelled with different dyes, and hybridized to a microarray. The DNA probes of gene A and gene C on the microarray will hybridize to the samples generated from both conditions. However, the cDNA sample of gene B will be present at reduced levels only in the test condition. By comparing the signal intensity from the two conditions for each probe, genes involved in growth or survival of the test condition can be identified. (C) Reproducibility of M-TraM. Log-log plot of the microarray signal intensities from two independent experiments of mutant libraries grown under non-selective conditions in BHI broth.
    Figure Legend Snippet: Schematic diagram and reproducibility of M-TraM. (A) Schematic overview of the M-TraM screening. In yellow: inverted terminal repeats (ITRs) of the himar1 transposon with outward-facing T7 promoters; in blue: the gentamicin resistance gene in the transposon. Genomic DNA is isolated from the E. faecium mutant library. DNA is digested with the restriction enzyme Alu I, and the DNA fragments are circularized by self-ligation. The transposon-chromosome junction together with an ITR and a T7 promoter is amplified by PCR with primers (blue arrow) that hybridize to the transposon. To eliminate foreign DNA fragments that ligated into the circularized DNA of transposon-chromosome junctions, the PCR products were re-digested with Alu I. The purified DNA fragments are used as template in the in vitro transcription reaction. The resulting RNA products are reverse transcribed into cDNA. After labelling, the cDNA is used for microarray hybridization. (B) Schematic overview of the screening strategy to identify conditionally essential genes by M-TraM. A chromosomal region encompassing three genes (A, B, and C) from three different mutants (1, 2, and 3) is shown. Each mutant carries a single transposon insertion (blue) that disrupts the function of the gene. Mutant libraries are grown in a control condition ( e.g. , BHI) and a test condition ( e.g. , in the presence of ampicillin). All the three genes are non-essential for growth in the control condition. Gene B is required only for the test condition, so mutant 2 exhibits attenuated growth or poorer survival only in the test condition, and will consequently be reduced or be entirely lost from this library (indicated by light shading). M-TraM samples are generated from the two conditions, labelled with different dyes, and hybridized to a microarray. The DNA probes of gene A and gene C on the microarray will hybridize to the samples generated from both conditions. However, the cDNA sample of gene B will be present at reduced levels only in the test condition. By comparing the signal intensity from the two conditions for each probe, genes involved in growth or survival of the test condition can be identified. (C) Reproducibility of M-TraM. Log-log plot of the microarray signal intensities from two independent experiments of mutant libraries grown under non-selective conditions in BHI broth.

    Techniques Used: Isolation, Mutagenesis, Ligation, Amplification, Polymerase Chain Reaction, Purification, In Vitro, Microarray, Hybridization, Generated

    Footprinting analysis of the transposon mutant library. (A) Schematic overview of the transposon footprinting strategy. PCR is performed using a gene specific primer and a primer corresponding to the transposon sequence. (B) Agarose gel electrophoresis of transposon footprinting on the essential gene ddl (lane 1), and the non-essential genes nox and esp (lane 2 and 3, respectively). Each band represents a PCR product of a different size, corresponding to a transposon insertion in a different position. The red box represents the product size range expected for transposon insertions within the essential ddl gene.
    Figure Legend Snippet: Footprinting analysis of the transposon mutant library. (A) Schematic overview of the transposon footprinting strategy. PCR is performed using a gene specific primer and a primer corresponding to the transposon sequence. (B) Agarose gel electrophoresis of transposon footprinting on the essential gene ddl (lane 1), and the non-essential genes nox and esp (lane 2 and 3, respectively). Each band represents a PCR product of a different size, corresponding to a transposon insertion in a different position. The red box represents the product size range expected for transposon insertions within the essential ddl gene.

    Techniques Used: Footprinting, Mutagenesis, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, End-sequence Profiling

    10) Product Images from "Equine Arteritis Virus Does Not Induce Interferon Production in Equine Endothelial Cells: Identification of Nonstructural Protein 1 as a Main Interferon Antagonist"

    Article Title: Equine Arteritis Virus Does Not Induce Interferon Production in Equine Endothelial Cells: Identification of Nonstructural Protein 1 as a Main Interferon Antagonist

    Journal: BioMed Research International

    doi: 10.1155/2014/420658

    Inhibition of type I IFN production after EAV infection. (a) Expression levels of IFN- β mRNA in EAV infected cells. EECs were mock-infected or infected with EAV VBS at an m.o.i. of 5 for 8 h. Subsequently, cells were infected with Sendai virus (SeV; 100 HAU/mL) for 3 h or 6 h. Total RNA was isolated and real-time RT-PCR was performed for the detection of equine IFN- β . Bar graph showing relative quantitation (RQ) values of IFN- β mRNA expression from three independent experiments are shown. (b) VSV bioassay for IFN production. EECs were mock-infected or infected with EAV VBS at an m.o.i. of 1 for 24 h. SeV was used as an IFN stimulator. Cell culture supernatants were collected and UV-irradiated for 30 min prior to use in the assay. MDBK cells were grown in 96-well plates and incubated with 2-fold dilution series of the supernatant up to 1/32. After 24 h incubation, cells were infected with VSV-GFP at an m.o.i. of 0.1, and 18 h after infection GFP expression was assessed by fluorescence microscopy. Each dilution was tested in duplicate.
    Figure Legend Snippet: Inhibition of type I IFN production after EAV infection. (a) Expression levels of IFN- β mRNA in EAV infected cells. EECs were mock-infected or infected with EAV VBS at an m.o.i. of 5 for 8 h. Subsequently, cells were infected with Sendai virus (SeV; 100 HAU/mL) for 3 h or 6 h. Total RNA was isolated and real-time RT-PCR was performed for the detection of equine IFN- β . Bar graph showing relative quantitation (RQ) values of IFN- β mRNA expression from three independent experiments are shown. (b) VSV bioassay for IFN production. EECs were mock-infected or infected with EAV VBS at an m.o.i. of 1 for 24 h. SeV was used as an IFN stimulator. Cell culture supernatants were collected and UV-irradiated for 30 min prior to use in the assay. MDBK cells were grown in 96-well plates and incubated with 2-fold dilution series of the supernatant up to 1/32. After 24 h incubation, cells were infected with VSV-GFP at an m.o.i. of 0.1, and 18 h after infection GFP expression was assessed by fluorescence microscopy. Each dilution was tested in duplicate.

    Techniques Used: Inhibition, Infection, Expressing, Isolation, Quantitative RT-PCR, Quantitation Assay, Cell Culture, Irradiation, Incubation, Fluorescence, Microscopy

    11) Product Images from "Tissue-specific splicing regulator Fox-1 induces exon skipping by interfering E complex formation on the downstream intron of human F1? gene"

    Article Title: Tissue-specific splicing regulator Fox-1 induces exon skipping by interfering E complex formation on the downstream intron of human F1? gene

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm569

    Mouse Fox-1 induces exon 9 skipping of hF1γ pre-mRNA by repressing the splicing of intron 9 via binding to GCAUG element in intron 8. ( A ) Analysis of intron 8 splicing in CV-1 cells. The Ex8-9 (lanes 1–3) and Ex8-9 mt mini-genes (lanes 4–6) were co-transfected with pCS2+MT vector (lanes 1 and 4), mFox-1 (lanes 2 and 5), F-A (lanes 3 and 6). The splicing products were analyzed by RT-PCR. A GFP plasmid was cotransfected as an internal reference for transfection efficiency, RNA recovery and loading. The positions of spliced products and GFP are indicated on the right. Average percentage and SD of splicing efficiency are shown at the bottom of each lane. ( B ) Analysis of intron 9 splicing in CV-1 cells. Transfection analyses of the Ex9-10 (lanes 1–3) and Ex9-10 mt mini-genes (lanes 4–6) with pCS2+MT vector (lanes 1 and 4), mFox-1 (lanes 2 and 5), F-A (lanes 3 and 6). The positions of spliced products and GFP are indicated on the right. Average percentage and SD of splicing efficiency are shown at the bottom of each lane. ( C ) Transfection analyses of the BPmt (lanes 1–3) and 5′SSmt (lanes 4–6) mini-genes with pCS2+MT vector (lanes 1 and 4), mFox-1 (lanes 2 and 5), F-A (lanes 3 and 6). Schematic representation of mini-genes is shown on the left of each panel. Open and closed circles show the GCAUG element and its mutated element, CGAUG, respectively. Open and closed triangles show branch point (BP) and its mutated site, respectively. A cross represents a mutated 5′ splice site in intron 9. Sequences of these elements are shown at the bottom. A bold letter represents the branch point nucleotide.
    Figure Legend Snippet: Mouse Fox-1 induces exon 9 skipping of hF1γ pre-mRNA by repressing the splicing of intron 9 via binding to GCAUG element in intron 8. ( A ) Analysis of intron 8 splicing in CV-1 cells. The Ex8-9 (lanes 1–3) and Ex8-9 mt mini-genes (lanes 4–6) were co-transfected with pCS2+MT vector (lanes 1 and 4), mFox-1 (lanes 2 and 5), F-A (lanes 3 and 6). The splicing products were analyzed by RT-PCR. A GFP plasmid was cotransfected as an internal reference for transfection efficiency, RNA recovery and loading. The positions of spliced products and GFP are indicated on the right. Average percentage and SD of splicing efficiency are shown at the bottom of each lane. ( B ) Analysis of intron 9 splicing in CV-1 cells. Transfection analyses of the Ex9-10 (lanes 1–3) and Ex9-10 mt mini-genes (lanes 4–6) with pCS2+MT vector (lanes 1 and 4), mFox-1 (lanes 2 and 5), F-A (lanes 3 and 6). The positions of spliced products and GFP are indicated on the right. Average percentage and SD of splicing efficiency are shown at the bottom of each lane. ( C ) Transfection analyses of the BPmt (lanes 1–3) and 5′SSmt (lanes 4–6) mini-genes with pCS2+MT vector (lanes 1 and 4), mFox-1 (lanes 2 and 5), F-A (lanes 3 and 6). Schematic representation of mini-genes is shown on the left of each panel. Open and closed circles show the GCAUG element and its mutated element, CGAUG, respectively. Open and closed triangles show branch point (BP) and its mutated site, respectively. A cross represents a mutated 5′ splice site in intron 9. Sequences of these elements are shown at the bottom. A bold letter represents the branch point nucleotide.

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

    Mouse Fox-1 induces exon 9 skipping of hF1γ pre-mRNA via binding to GCAUG element. ( A ) The schematic representation of various hF1γ mini-genes. The Fox-1-binding sequence GCAUG and its mutated sequence CGAUG are shown as open and closed circles, respectively. ( B ) Transfection assays of various hF1γ mini-genes into CV-1 cells. The hF1γL (lanes 1–3), hF1γS (lanes 4–6), hF1γSmt (lanes 7–9) and hF1γSmt+3GCAUG mini-genes (lanes 10–12) were co-expressed with pCS2+MT vector (lanes 1, 4, 7, 10), Fox-1 (lanes 2, 5, 8, 11) and F-A mutant (lanes 3, 6, 9, 12). Splicing products were analyzed by RT-PCR. Splicing products are schematically shown on the right. All experiments were performed more than three times. Average percentage and SD of exon 9 exclusion are shown at the bottom of each lane. ( C ) Upper panel shows western blotting of cell extracts to detect Fox-1 proteins: Mock, Fox-1 and Fox-1 F-A, expressed from the pCS2+MT vector using the anti-Myc antibody. The positions of molecular size markers are shown on the right. Lower panel shows western blotting of the same cell extracts with anti-U2AF antibody as a loading control.
    Figure Legend Snippet: Mouse Fox-1 induces exon 9 skipping of hF1γ pre-mRNA via binding to GCAUG element. ( A ) The schematic representation of various hF1γ mini-genes. The Fox-1-binding sequence GCAUG and its mutated sequence CGAUG are shown as open and closed circles, respectively. ( B ) Transfection assays of various hF1γ mini-genes into CV-1 cells. The hF1γL (lanes 1–3), hF1γS (lanes 4–6), hF1γSmt (lanes 7–9) and hF1γSmt+3GCAUG mini-genes (lanes 10–12) were co-expressed with pCS2+MT vector (lanes 1, 4, 7, 10), Fox-1 (lanes 2, 5, 8, 11) and F-A mutant (lanes 3, 6, 9, 12). Splicing products were analyzed by RT-PCR. Splicing products are schematically shown on the right. All experiments were performed more than three times. Average percentage and SD of exon 9 exclusion are shown at the bottom of each lane. ( C ) Upper panel shows western blotting of cell extracts to detect Fox-1 proteins: Mock, Fox-1 and Fox-1 F-A, expressed from the pCS2+MT vector using the anti-Myc antibody. The positions of molecular size markers are shown on the right. Lower panel shows western blotting of the same cell extracts with anti-U2AF antibody as a loading control.

    Techniques Used: Binding Assay, Sequencing, Transfection, Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Western Blot

    12) Product Images from "TRAIL treatment provokes mutations in surviving cells"

    Article Title: TRAIL treatment provokes mutations in surviving cells

    Journal: Oncogene

    doi: 10.1038/onc.2010.242

    CrmA expression prevents the formation of 6-TG-resistant mouse embryo fibroblast (MEF) colonies following TRAIL treatment. MEF cells were stably transfected with empty vector (pEF), a wild-type FLAG-tagged crmA expression plasmid or a loss-of-function FLAG-crmA expression plasmid. Genomic DNA from pEF clones was amplified using PCR ( a ). Products of reactions containing either no template or 100 ng of plasmid are denoted ‘pos cont' and ‘neg cont' respectively. CrmA expression was analyzed using anti-FLAG immunoblotting, relative to a GAPDH loading control ( b ). ( c ) The phenotypes of crmA wild-type and mutant clones were tested by transiently transfecting the clones with empty vector (pEF), crmA and/or caspase-8 expression plasmids (90% of transfected DNA), along with a β-galactosidase expression plasmid (10%). The transfectants were stained with Xgal and the blue (transfected) cells were scored visually for morphological characteristics of apoptosis. The proportions of each transiently transfected plasmid are indicated under the graph, as are the stable clones into which this DNA was introduced. Clonogenic survival of each clone was monitored following 1 h exposure to cross-linked TRAIL ( d ) or cisplatin ( f ). Parental MEF cells and the indicated stable cell lines were treated with cross-linked TRAIL( e ) or cisplatin ( g ) for 1 h then subjected to the HPRT mutational assay, and the 6-TG-resistant clones that emerged were counted.
    Figure Legend Snippet: CrmA expression prevents the formation of 6-TG-resistant mouse embryo fibroblast (MEF) colonies following TRAIL treatment. MEF cells were stably transfected with empty vector (pEF), a wild-type FLAG-tagged crmA expression plasmid or a loss-of-function FLAG-crmA expression plasmid. Genomic DNA from pEF clones was amplified using PCR ( a ). Products of reactions containing either no template or 100 ng of plasmid are denoted ‘pos cont' and ‘neg cont' respectively. CrmA expression was analyzed using anti-FLAG immunoblotting, relative to a GAPDH loading control ( b ). ( c ) The phenotypes of crmA wild-type and mutant clones were tested by transiently transfecting the clones with empty vector (pEF), crmA and/or caspase-8 expression plasmids (90% of transfected DNA), along with a β-galactosidase expression plasmid (10%). The transfectants were stained with Xgal and the blue (transfected) cells were scored visually for morphological characteristics of apoptosis. The proportions of each transiently transfected plasmid are indicated under the graph, as are the stable clones into which this DNA was introduced. Clonogenic survival of each clone was monitored following 1 h exposure to cross-linked TRAIL ( d ) or cisplatin ( f ). Parental MEF cells and the indicated stable cell lines were treated with cross-linked TRAIL( e ) or cisplatin ( g ) for 1 h then subjected to the HPRT mutational assay, and the 6-TG-resistant clones that emerged were counted.

    Techniques Used: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Clone Assay, Amplification, Polymerase Chain Reaction, Mutagenesis, Staining

    13) Product Images from "Exposure to 17β-Oestradiol Induces Oxidative Stress in the Non-Oestrogen Receptor Invertebrate Species Eisenia fetida"

    Article Title: Exposure to 17β-Oestradiol Induces Oxidative Stress in the Non-Oestrogen Receptor Invertebrate Species Eisenia fetida

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145426

    Influence of E 2 exposure (100 μg/kg) on the expression of mRNA encoding selected antioxidant molecules. ( A ) MT , ( B ) GPx and ( C ) pcs genes in E . fetida , collected in the 1 st ; 3 rd ; 5 th and 8 th weeks of the experiment. For evaluation, the corresponding molecular-weight bands of amplicons coinciding with the values shown in Table 1 were obtained using RT-PCR. The fluorescence intensities of the bands, obtained using ethidium bromide staining, were transformed into numerical forms, and expression was normalized to β-actin expression from the same cDNA template. The values are presented as the means of three independent replicates ( n = 3). The vertical bars indicate standard error. The asterisks indicate significant differences ( p
    Figure Legend Snippet: Influence of E 2 exposure (100 μg/kg) on the expression of mRNA encoding selected antioxidant molecules. ( A ) MT , ( B ) GPx and ( C ) pcs genes in E . fetida , collected in the 1 st ; 3 rd ; 5 th and 8 th weeks of the experiment. For evaluation, the corresponding molecular-weight bands of amplicons coinciding with the values shown in Table 1 were obtained using RT-PCR. The fluorescence intensities of the bands, obtained using ethidium bromide staining, were transformed into numerical forms, and expression was normalized to β-actin expression from the same cDNA template. The values are presented as the means of three independent replicates ( n = 3). The vertical bars indicate standard error. The asterisks indicate significant differences ( p

    Techniques Used: Expressing, Molecular Weight, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Staining, Transformation Assay

    14) Product Images from "Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina"

    Article Title: Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017647

    CLC H + /Cl − antiporters are expressed in chicken neurons. A , RT-PCR amplification of ClCs 3–7 mRNA from a mixed population of cultured chick retinal neurons. Two primer pairs for each transporter amplified single products of the expected size (in kb). B , Western blots of chicken, rat, and mouse brain protein probed with antibodies specific for mammalian ClCs 3–7 reveal labeling of bands at the expected molecular weights for each protein. CB, chicken brain; RB, rat brain; MB, mouse brain.
    Figure Legend Snippet: CLC H + /Cl − antiporters are expressed in chicken neurons. A , RT-PCR amplification of ClCs 3–7 mRNA from a mixed population of cultured chick retinal neurons. Two primer pairs for each transporter amplified single products of the expected size (in kb). B , Western blots of chicken, rat, and mouse brain protein probed with antibodies specific for mammalian ClCs 3–7 reveal labeling of bands at the expected molecular weights for each protein. CB, chicken brain; RB, rat brain; MB, mouse brain.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Cell Culture, Western Blot, Labeling

    15) Product Images from "CD3-Positive B Cells: A Storage-Dependent Phenomenon"

    Article Title: CD3-Positive B Cells: A Storage-Dependent Phenomenon

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0110138

    Detection of CD3 on B cell surfaces is a result of cell number- dependent T-B cell interactions. Semiquantitative RT-PCR for CD3 was performed using RNA isolated from 7×10 5 MACS-purified CD20 + B cells and a CD3-expressing T cell line (TCL). (A) Endogenous expression of CD3 was only found in the TCL, which served as a positive control, but neither in CD3 - CD20 + nor in CD3 low CD20 + B cells. β-actin was used as loading control. (B) The appearance of CD3 low CD20 + B cells was demonstrated after co-culture of MACS-purified CD4 + T cells and CD20 + B cells in different ratios overnight (oN) at 4°C. Storage of CD20 + B cells alone under the same conditions was not associated with an induction of the CD3 low CD20 + B cell population, pointing at the need for T cells. Furthermore, the quantity of CD3 low CD20 + B cells was dependent on the number of CD4 + T cells, i.e. increasing T-B cell ratios caused elevated numbers of CD3 low CD20 + B cells. Shown are the results of up to three independent experiments.
    Figure Legend Snippet: Detection of CD3 on B cell surfaces is a result of cell number- dependent T-B cell interactions. Semiquantitative RT-PCR for CD3 was performed using RNA isolated from 7×10 5 MACS-purified CD20 + B cells and a CD3-expressing T cell line (TCL). (A) Endogenous expression of CD3 was only found in the TCL, which served as a positive control, but neither in CD3 - CD20 + nor in CD3 low CD20 + B cells. β-actin was used as loading control. (B) The appearance of CD3 low CD20 + B cells was demonstrated after co-culture of MACS-purified CD4 + T cells and CD20 + B cells in different ratios overnight (oN) at 4°C. Storage of CD20 + B cells alone under the same conditions was not associated with an induction of the CD3 low CD20 + B cell population, pointing at the need for T cells. Furthermore, the quantity of CD3 low CD20 + B cells was dependent on the number of CD4 + T cells, i.e. increasing T-B cell ratios caused elevated numbers of CD3 low CD20 + B cells. Shown are the results of up to three independent experiments.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Magnetic Cell Separation, Purification, Expressing, Positive Control, Co-Culture Assay

    16) Product Images from "Deletion Mutants of VPg Reveal New Cytopathology Determinants in a Picornavirus"

    Article Title: Deletion Mutants of VPg Reveal New Cytopathology Determinants in a Picornavirus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010735

    Replication of mutant FMDVs in BHK–21 cells. A–C , Western blot assays for the specific detection of FMDV proteins using monoclonal antibody (MAb) 1C8 specific for 2C (panel A), a polyclonal antibody (PAb) specific for 3D (panel B), and MAb 2D2 specific for 3C (panel C). A total of 10 6 BHK–21 cells were mock–electroporated (BHK lane) or electroporated with either 2 µg of WT RNA or 8 µg of the mutant transcript indicated in each lane. A 4–fold excess of transcript from mutant plasmids was required to reach a comparable level of viral proteins for WT and mutant RNAs. At 4 hours post–electroporation cells were collected in lysis buffer, subjected to SDS–PAGE, and then transferred to a nitrocellulose membrane. The MAbs and the PAb have been previously described [62] . Molecular size markers were run in parallel and their position is indicated on the left. The positions of viral proteins 2BC, 2C, P3, 3CD, 3BC and 3C, determined with specific MAbs, are indicated [62] . Note that precursor P3 (3ABCD, highlighted with an asterisk) displayed a higher mobility than WT in all the mutant transcripts, consistent with a decrease of the number of VPg copies. The right panel in C, (lanes V3, WT and V19–4) correspond to the analysis of cells collected at 5 h instead of 4 h post–transfection, and is included here because of easier band identification. D , Average number of viral RNA molecules per cell (quantitated both inside the cells and in the culture medium) at 72 hours post–transfection of 2×10 6 BHK–21 cells with 100 ng of the indicated RNA transcripts. The cells and supernatants were harvested at 72 hours post–transfection, and the number of genomic RNA molecules was calculated by quantitative real–time RT–PCR, as described in Materials and Methods (limit of detection 8 viral RNA molecules/cell, indicated as a dashed line). E , Viral titre (PFU/ml) in the transfected cultures (both the supernatant and cells subjected to freeze–thawing) described in D. Plaque assays were performed as described in Materials and Methods . Plaque development was permitted for 48h (limit of detection 5 PFU/ml, indicated as a dashed line). Measurements in D, E were carried out in triplicate and standard deviations are given.
    Figure Legend Snippet: Replication of mutant FMDVs in BHK–21 cells. A–C , Western blot assays for the specific detection of FMDV proteins using monoclonal antibody (MAb) 1C8 specific for 2C (panel A), a polyclonal antibody (PAb) specific for 3D (panel B), and MAb 2D2 specific for 3C (panel C). A total of 10 6 BHK–21 cells were mock–electroporated (BHK lane) or electroporated with either 2 µg of WT RNA or 8 µg of the mutant transcript indicated in each lane. A 4–fold excess of transcript from mutant plasmids was required to reach a comparable level of viral proteins for WT and mutant RNAs. At 4 hours post–electroporation cells were collected in lysis buffer, subjected to SDS–PAGE, and then transferred to a nitrocellulose membrane. The MAbs and the PAb have been previously described [62] . Molecular size markers were run in parallel and their position is indicated on the left. The positions of viral proteins 2BC, 2C, P3, 3CD, 3BC and 3C, determined with specific MAbs, are indicated [62] . Note that precursor P3 (3ABCD, highlighted with an asterisk) displayed a higher mobility than WT in all the mutant transcripts, consistent with a decrease of the number of VPg copies. The right panel in C, (lanes V3, WT and V19–4) correspond to the analysis of cells collected at 5 h instead of 4 h post–transfection, and is included here because of easier band identification. D , Average number of viral RNA molecules per cell (quantitated both inside the cells and in the culture medium) at 72 hours post–transfection of 2×10 6 BHK–21 cells with 100 ng of the indicated RNA transcripts. The cells and supernatants were harvested at 72 hours post–transfection, and the number of genomic RNA molecules was calculated by quantitative real–time RT–PCR, as described in Materials and Methods (limit of detection 8 viral RNA molecules/cell, indicated as a dashed line). E , Viral titre (PFU/ml) in the transfected cultures (both the supernatant and cells subjected to freeze–thawing) described in D. Plaque assays were performed as described in Materials and Methods . Plaque development was permitted for 48h (limit of detection 5 PFU/ml, indicated as a dashed line). Measurements in D, E were carried out in triplicate and standard deviations are given.

    Techniques Used: Mutagenesis, Western Blot, Electroporation, Lysis, SDS Page, Transfection, Quantitative RT-PCR

    Effect of replacement R55W in 2C in the release of FMDV RNA from cells. 10 6 BHK–21 cells were electroporated with 8 µg of either V19–4, V3, V15–9 or WT RNAs. Then the cells were transferred to M24–wells. A , Total viral RNA (intracellular and extracellular) and extracellular RNA were quantified by real–time RT–PCR, at the indicated hours post-electroporation (hpe). The amount of FMDV RNA was normalised to the number of cells seeded in the corresponding wells. The viral RNA (total and extracellular) measured just after electroporation was subtracted from each corresponding value. Note that RNAs encoding R55W in 2C are significantly more efficient in promoting release of viral RNA from cells at 6 hours post–transfection than those expressing wild type 2C (p
    Figure Legend Snippet: Effect of replacement R55W in 2C in the release of FMDV RNA from cells. 10 6 BHK–21 cells were electroporated with 8 µg of either V19–4, V3, V15–9 or WT RNAs. Then the cells were transferred to M24–wells. A , Total viral RNA (intracellular and extracellular) and extracellular RNA were quantified by real–time RT–PCR, at the indicated hours post-electroporation (hpe). The amount of FMDV RNA was normalised to the number of cells seeded in the corresponding wells. The viral RNA (total and extracellular) measured just after electroporation was subtracted from each corresponding value. Note that RNAs encoding R55W in 2C are significantly more efficient in promoting release of viral RNA from cells at 6 hours post–transfection than those expressing wild type 2C (p

    Techniques Used: Quantitative RT-PCR, Electroporation, Transfection, Expressing

    17) Product Images from "Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2"

    Article Title: Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.00967

    Detection of DM2 mutation at DNA, expression of CNBP gene and RNA level during differentiation. (A) Percentages of nuclear foci (1–4 or > 5) in hiPSCs and NP showing the increase of foci number along their differentiation process. (B) LR-PCR followed by hybridization with a (CTG) 5 -radioactively labeled probe on DNA extracted from DM2 hiPSCs and NPs, arrows indicated CNBP normal and expanded alleles. (C) RT-qPCR assay for CNBP expression in DM2 hiPSCs and NPs. β-actin is used as reference gene.
    Figure Legend Snippet: Detection of DM2 mutation at DNA, expression of CNBP gene and RNA level during differentiation. (A) Percentages of nuclear foci (1–4 or > 5) in hiPSCs and NP showing the increase of foci number along their differentiation process. (B) LR-PCR followed by hybridization with a (CTG) 5 -radioactively labeled probe on DNA extracted from DM2 hiPSCs and NPs, arrows indicated CNBP normal and expanded alleles. (C) RT-qPCR assay for CNBP expression in DM2 hiPSCs and NPs. β-actin is used as reference gene.

    Techniques Used: Mutagenesis, Expressing, Polymerase Chain Reaction, Hybridization, CTG Assay, Labeling, Quantitative RT-PCR

    18) Product Images from "Phosphoglucose isomerase/autocrine motility factor mediates epithelial and mesenchymal phenotype conversions in breast cancer"

    Article Title: Phosphoglucose isomerase/autocrine motility factor mediates epithelial and mesenchymal phenotype conversions in breast cancer

    Journal:

    doi: 10.1158/0008-5472.CAN-09-0488

    Mechanisms controlling E-cadherin and ß-catenin expression in PGI/AMF-overexpressed cells. A, cells were analyzed by RT-PCR for E-cadherin, ß-catenin and ß-actin expression. Representative results of three different experiments
    Figure Legend Snippet: Mechanisms controlling E-cadherin and ß-catenin expression in PGI/AMF-overexpressed cells. A, cells were analyzed by RT-PCR for E-cadherin, ß-catenin and ß-actin expression. Representative results of three different experiments

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    19) Product Images from "PCR amplification of a long rDNA segment with one primer pair in agriculturally important nematodes"

    Article Title: PCR amplification of a long rDNA segment with one primer pair in agriculturally important nematodes

    Journal: Journal of Nematology

    doi: 10.21307/jofnem-2019-026

    PCR Amplification of 3.3 to 4.2 kb of rDNA from agriculturally important nematodes. M: DNA markers; 1: Heterodera orientalis 104F80; 2: Xiphinema sp. 104F83; 3: Hoplolaimus sp. 104G35; 4: Helicotylenchus sp. 104G36; 5: Meloidogyne incognita Me47; 6: Pratylenchus scribneri Pr1 and 7: Negative control.
    Figure Legend Snippet: PCR Amplification of 3.3 to 4.2 kb of rDNA from agriculturally important nematodes. M: DNA markers; 1: Heterodera orientalis 104F80; 2: Xiphinema sp. 104F83; 3: Hoplolaimus sp. 104G35; 4: Helicotylenchus sp. 104G36; 5: Meloidogyne incognita Me47; 6: Pratylenchus scribneri Pr1 and 7: Negative control.

    Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control

    20) Product Images from "Autoregulation of the MisR/MisS Two-Component Signal Transduction System in Neisseria meningitidis †"

    Article Title: Autoregulation of the MisR/MisS Two-Component Signal Transduction System in Neisseria meningitidis †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00264-06

    EMSA of MisR- misR promoter interaction. (A) A 608-bp PCR product (∼5 fmol) of a DNA fragment containing the misR promoter region (−504 to +104) was 32 P-labeled by T4 kinase, mixed with increasing amounts of MisR∼P (left) and MisR (right) for 20 min at 30°C and then subjected to gel electrophoresis. MisR∼P was generated by incubation with 50 mM acetyl phosphate for 30 min at 37°C. The amounts of MisR protein are 0, 34, 68, 136, 204, 272, and 340 pmol. (B) Competition EMSA. 32 P-labeled misR probe was mixed with the indicated amounts of either specific or nonspecific DNA, incubated with MisR∼P (102 pmol), and analyzed as described above.
    Figure Legend Snippet: EMSA of MisR- misR promoter interaction. (A) A 608-bp PCR product (∼5 fmol) of a DNA fragment containing the misR promoter region (−504 to +104) was 32 P-labeled by T4 kinase, mixed with increasing amounts of MisR∼P (left) and MisR (right) for 20 min at 30°C and then subjected to gel electrophoresis. MisR∼P was generated by incubation with 50 mM acetyl phosphate for 30 min at 37°C. The amounts of MisR protein are 0, 34, 68, 136, 204, 272, and 340 pmol. (B) Competition EMSA. 32 P-labeled misR probe was mixed with the indicated amounts of either specific or nonspecific DNA, incubated with MisR∼P (102 pmol), and analyzed as described above.

    Techniques Used: Polymerase Chain Reaction, Labeling, Nucleic Acid Electrophoresis, Generated, Incubation

    21) Product Images from "Cerebral organoids model human brain development and microcephaly"

    Article Title: Cerebral organoids model human brain development and microcephaly

    Journal: Nature

    doi: 10.1038/nature12517

    Human cerebral organoids recapitulate various brain region identities a, RT-PCR for forebrain markers (BF1 and Six3) and hindbrain markers (Krox20 and Isl1) at 12, 16 and 20 days of differentiation. Human fetal brain cDNA was used as positive control. b, Immunohistochemistry in serial sections for the forebrain marker Pax6 (red, first panel) and the hindbrain markers Krox20 (green, first panel) and Pax2 (red, second panel) at 16 days of differentiation. Note the juxtaposition reminiscent of the mid-hindbrain boundary (arrows). DAPI marks nuclei (blue). c,-i, Staining for various brain region identities: forebrain, FoxG1 ( c ); dorsal cortex, Emx1 ( d ); prefrontal cortex (note the discrete boundary, arrow), Auts2 ( e ); hippocampus, Nrp2, Fzd9, Prox1 ( f ); ventral forebrain, Nkx2.1 ( g ) and choroid plexus, TTR ( i ). g, Staining for adjacent ventral (arrow) and dorsal (Pax6, arrowhead) forebrain and for calretinin (green) in a serial section revealing cortical interneurons in the ventral region (arrow). Calretinin interneurons within dorsal cortex ( h ) exhibit typical morphology of tangential migration (arrows). j, Hematoxylin-eosin staining of retinal tissue exhibiting stereotypical layering: retinal pigment epithelium (RPE), outer nuclear layer (ONL) and inner nuclear layer (INL). Scale bars: 100 μm.
    Figure Legend Snippet: Human cerebral organoids recapitulate various brain region identities a, RT-PCR for forebrain markers (BF1 and Six3) and hindbrain markers (Krox20 and Isl1) at 12, 16 and 20 days of differentiation. Human fetal brain cDNA was used as positive control. b, Immunohistochemistry in serial sections for the forebrain marker Pax6 (red, first panel) and the hindbrain markers Krox20 (green, first panel) and Pax2 (red, second panel) at 16 days of differentiation. Note the juxtaposition reminiscent of the mid-hindbrain boundary (arrows). DAPI marks nuclei (blue). c,-i, Staining for various brain region identities: forebrain, FoxG1 ( c ); dorsal cortex, Emx1 ( d ); prefrontal cortex (note the discrete boundary, arrow), Auts2 ( e ); hippocampus, Nrp2, Fzd9, Prox1 ( f ); ventral forebrain, Nkx2.1 ( g ) and choroid plexus, TTR ( i ). g, Staining for adjacent ventral (arrow) and dorsal (Pax6, arrowhead) forebrain and for calretinin (green) in a serial section revealing cortical interneurons in the ventral region (arrow). Calretinin interneurons within dorsal cortex ( h ) exhibit typical morphology of tangential migration (arrows). j, Hematoxylin-eosin staining of retinal tissue exhibiting stereotypical layering: retinal pigment epithelium (RPE), outer nuclear layer (ONL) and inner nuclear layer (INL). Scale bars: 100 μm.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Positive Control, Immunohistochemistry, Marker, Staining, Migration

    22) Product Images from "Endoplasmic reticulum stress induces PRNP prion protein gene expression in breast cancer"

    Article Title: Endoplasmic reticulum stress induces PRNP prion protein gene expression in breast cancer

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr3398

    sXBP1 is involved in PRNP gene expression in MCF-7 and MDA-MB-231 cells . (A) Western blot analyses of PrP with the 3F4 antibody, cleaved ATF6α (ΔATF6α), phosphorylated eIF2α (peIF2α), total eIF2α, and β-actin. Lower panels represent XBP1, spliced XBP1 (sXBP1) and β-actin amplified by RT-PCR. Protein or mRNA extracts were from MCF-7 cells treated with ER stressors for 0 to 6 hrs. The increase of PrP and ΔATF6α levels compared to β-actin levels and the ratios of peIF2α/eIF2α and sXBP1/XBP1 are indicated. (B) MCF-7 cells transfected with siATF6α or siXBP1 and proteins immunoblotted with 3F4 PrP and β-actin antibodies. RT-PCR shows levels of ATF6α and XBP1 mRNAs. (C) Western blot analyses of PrP, HA tag and β-actin in protein extracts from MCF-7 cells transfected for 6 hrs with pCGN-IRES-EGFP (Ctl), pCGN-HA-sXBP1-IRES-EGFP, and pCGN-HA-ATF4-IRES-EGFP constructs. (D) ChIP assays performed on DMSO (Ctl)- or BFA-treated MCF-7 cells with IgG control, XBP1 or ATF6α antibodies. PCR amplification of PRNP and β-actin gene promoters ( ACTB ) was done on immunoprecipitated and non-immunoprecipitated (input) DNA. ( E ) Western blot of PrP (top panel) and β-actin (bottom panel) and ethidium stained agarose gel containing ATF6α and XBP1 amplicons from MDA-MB-231 or HS578T cells transfected with siCtl, siATF6α or siXBP1. NT indicates non-transfected, D indicates the Dharmacon siRNAs and SC indicates the Santa Cruz siRNAs. ( F) Levels of PRNP mRNA detected by qRT-PCR in siATF6α or siXBP1-transfected cells.
    Figure Legend Snippet: sXBP1 is involved in PRNP gene expression in MCF-7 and MDA-MB-231 cells . (A) Western blot analyses of PrP with the 3F4 antibody, cleaved ATF6α (ΔATF6α), phosphorylated eIF2α (peIF2α), total eIF2α, and β-actin. Lower panels represent XBP1, spliced XBP1 (sXBP1) and β-actin amplified by RT-PCR. Protein or mRNA extracts were from MCF-7 cells treated with ER stressors for 0 to 6 hrs. The increase of PrP and ΔATF6α levels compared to β-actin levels and the ratios of peIF2α/eIF2α and sXBP1/XBP1 are indicated. (B) MCF-7 cells transfected with siATF6α or siXBP1 and proteins immunoblotted with 3F4 PrP and β-actin antibodies. RT-PCR shows levels of ATF6α and XBP1 mRNAs. (C) Western blot analyses of PrP, HA tag and β-actin in protein extracts from MCF-7 cells transfected for 6 hrs with pCGN-IRES-EGFP (Ctl), pCGN-HA-sXBP1-IRES-EGFP, and pCGN-HA-ATF4-IRES-EGFP constructs. (D) ChIP assays performed on DMSO (Ctl)- or BFA-treated MCF-7 cells with IgG control, XBP1 or ATF6α antibodies. PCR amplification of PRNP and β-actin gene promoters ( ACTB ) was done on immunoprecipitated and non-immunoprecipitated (input) DNA. ( E ) Western blot of PrP (top panel) and β-actin (bottom panel) and ethidium stained agarose gel containing ATF6α and XBP1 amplicons from MDA-MB-231 or HS578T cells transfected with siCtl, siATF6α or siXBP1. NT indicates non-transfected, D indicates the Dharmacon siRNAs and SC indicates the Santa Cruz siRNAs. ( F) Levels of PRNP mRNA detected by qRT-PCR in siATF6α or siXBP1-transfected cells.

    Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot, Amplification, Reverse Transcription Polymerase Chain Reaction, Transfection, CTL Assay, Construct, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Staining, Agarose Gel Electrophoresis, Quantitative RT-PCR

    23) Product Images from "Identification of human thioredoxin as a novel IFN-gamma-induced factor: Mechanism of induction and its role in cytokine production"

    Article Title: Identification of human thioredoxin as a novel IFN-gamma-induced factor: Mechanism of induction and its role in cytokine production

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-9-64

    Regulation of thioredoxin gene expression in immune cell lines . A. Regulation of thioredoxin gene expression by cytokines in immune cell lines . Human lymphocytic T cell line, Jurkat ( Panel a ) and the promonocytic cell line, THP1 ( Panels b and c ), were maintained in complete RPMI media. Cells (5 × 10 6 ) were then treated with various cytokines (10 ng/ml IFN-γ, 10 ng/ml IL-4, 10000 u/ml IFN-α, and 10 ng/ml IL-2) as indicated for 24 h. The total RNA was then isolated and analyzed by Northern blot using a full-length cDNA probe of human thioredoxin. The membranes were stripped and reprobed for β-actin as an internal control. B. Effect of IFN-γ and mitogens on thioredoxin gene expression. Panel a : Jurkat T cells (2 × 10 6 ) were treated with IFN-γ (10 ng/ml), LPS (1 μg/ml), PMA (10 ng/ml) or PHA (2.5 μg/ml) in serum-free media for the indicated durations, after which RNAs were isolated and analyzed by RT-PCR using primers specific for thioredoxin. Panel b : THP1 monocytic cells (2 × 10 6 ) were treated with IFN-γ (10 ng/ml), LPS (1 μg/ml), or PHA (2.5 μg/ml) in the presence or absence of anti-IFN-γ Ab (10 μg/ml). The cells were then cultured for 24 h, after which the total RNA was isolated and analyzed by RT-PCR using primers specific for thioredoxin and IFN-γ.
    Figure Legend Snippet: Regulation of thioredoxin gene expression in immune cell lines . A. Regulation of thioredoxin gene expression by cytokines in immune cell lines . Human lymphocytic T cell line, Jurkat ( Panel a ) and the promonocytic cell line, THP1 ( Panels b and c ), were maintained in complete RPMI media. Cells (5 × 10 6 ) were then treated with various cytokines (10 ng/ml IFN-γ, 10 ng/ml IL-4, 10000 u/ml IFN-α, and 10 ng/ml IL-2) as indicated for 24 h. The total RNA was then isolated and analyzed by Northern blot using a full-length cDNA probe of human thioredoxin. The membranes were stripped and reprobed for β-actin as an internal control. B. Effect of IFN-γ and mitogens on thioredoxin gene expression. Panel a : Jurkat T cells (2 × 10 6 ) were treated with IFN-γ (10 ng/ml), LPS (1 μg/ml), PMA (10 ng/ml) or PHA (2.5 μg/ml) in serum-free media for the indicated durations, after which RNAs were isolated and analyzed by RT-PCR using primers specific for thioredoxin. Panel b : THP1 monocytic cells (2 × 10 6 ) were treated with IFN-γ (10 ng/ml), LPS (1 μg/ml), or PHA (2.5 μg/ml) in the presence or absence of anti-IFN-γ Ab (10 μg/ml). The cells were then cultured for 24 h, after which the total RNA was isolated and analyzed by RT-PCR using primers specific for thioredoxin and IFN-γ.

    Techniques Used: Expressing, Isolation, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    24) Product Images from "Absolute quantification of the budding yeast transcriptome by means of competitive PCR between genomic and complementary DNAs"

    Article Title: Absolute quantification of the budding yeast transcriptome by means of competitive PCR between genomic and complementary DNAs

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-9-574

    Calibration of GATC-PCR between genomic DNA and cDNA . (A) Competitive amplification of GCN4 between genomic DNA and cDNA. (B) Standard RNAs used for competitive PCR determination of mRNA copy number. (C) Comparison of absolute amounts of eight mRNAs determined by real-time PCR and GATC-PCR. For real-time PCR, we used each GSP for the first strand cDNA synthesis. The GATC-PCR data were calibrated by the competitive PCR quantification of GCN4 mRNA using the standard RNA set (Figure 2B, Table 3 ).
    Figure Legend Snippet: Calibration of GATC-PCR between genomic DNA and cDNA . (A) Competitive amplification of GCN4 between genomic DNA and cDNA. (B) Standard RNAs used for competitive PCR determination of mRNA copy number. (C) Comparison of absolute amounts of eight mRNAs determined by real-time PCR and GATC-PCR. For real-time PCR, we used each GSP for the first strand cDNA synthesis. The GATC-PCR data were calibrated by the competitive PCR quantification of GCN4 mRNA using the standard RNA set (Figure 2B, Table 3 ).

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

    Generalized Adaptor-Tagged Competitive PCR (GATC-PCR) . (A) Gene-specific primer (GSP)-dependent amplification from Y-shaped adaptor-tagged template. (B) An example of GATC-PCR. Genomic DNA and cDNA digested with Mbo I were ligated with adaptor A/C and B/C (Table 2 ), respectively, and used for GATC-PCR. The products of four assays (blue, green, red, and black) and a size standard (orange) were separated on ABI 3730 Genetic Analyzer. The fast- and slow-migrating peaks of each pair correspond to the signals from genomic DNA and cDNA, respectively. (C) Linearity of GATC-PCR from genomic DNA templates. Genomic DNAs extracted from the wild and gcn4 Δ cells were combined at appropriate ratios to prepare a series of genomic DNAs containing 0, 0.25, 0.5, 0.75, and 1 copy of GCN4 per haploid on average, digested with Mbo I, and ligated to the adaptors A/C and B/C (Table 2 ). Various combinations of the A/C- and B/C-tagged templates were mixed in a 1:1 ratio, while keeping the total amount equivalent to 3,000 haploid cells, and subjected to GATC-PCR using a GCN4 -specific primer. (D) Linearity of GATC-PCR from cDNA templates. An experiment similar to the one shown in (C) was conducted using cDNAs, instead of genomic DNA, prepared from the wild and gcn4 Δ cells.
    Figure Legend Snippet: Generalized Adaptor-Tagged Competitive PCR (GATC-PCR) . (A) Gene-specific primer (GSP)-dependent amplification from Y-shaped adaptor-tagged template. (B) An example of GATC-PCR. Genomic DNA and cDNA digested with Mbo I were ligated with adaptor A/C and B/C (Table 2 ), respectively, and used for GATC-PCR. The products of four assays (blue, green, red, and black) and a size standard (orange) were separated on ABI 3730 Genetic Analyzer. The fast- and slow-migrating peaks of each pair correspond to the signals from genomic DNA and cDNA, respectively. (C) Linearity of GATC-PCR from genomic DNA templates. Genomic DNAs extracted from the wild and gcn4 Δ cells were combined at appropriate ratios to prepare a series of genomic DNAs containing 0, 0.25, 0.5, 0.75, and 1 copy of GCN4 per haploid on average, digested with Mbo I, and ligated to the adaptors A/C and B/C (Table 2 ). Various combinations of the A/C- and B/C-tagged templates were mixed in a 1:1 ratio, while keeping the total amount equivalent to 3,000 haploid cells, and subjected to GATC-PCR using a GCN4 -specific primer. (D) Linearity of GATC-PCR from cDNA templates. An experiment similar to the one shown in (C) was conducted using cDNAs, instead of genomic DNA, prepared from the wild and gcn4 Δ cells.

    Techniques Used: Polymerase Chain Reaction, Amplification

    25) Product Images from "Developmentally regulated expression, alternative splicing and distinct sub-groupings in members of the Schistosoma mansoni venom allergen-like (SmVAL) gene family"

    Article Title: Developmentally regulated expression, alternative splicing and distinct sub-groupings in members of the Schistosoma mansoni venom allergen-like (SmVAL) gene family

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-9-89

    SmVAL transcription throughout the schistosome life cycle includes both developmental and constitutive patterns . Total RNA from indicated life-stages was obtained as described in Methods and utilized for real time quantitative PCR analysis to determine SmVAL1-13 transcript abundance. For each SmVAL transcript, a bar graph is displayed indicating relative abundance (compared to SmAT1) across the S. mansoni lifecycle. On the x -axis, each specific life-stage cDNA being tested is indicated. The y -axis represents the ratio of SmVAL gene expression relative to that of SmAT1 (reference gene). Data are presented as mean ratios (+/- standard deviation) from technical duplicates.
    Figure Legend Snippet: SmVAL transcription throughout the schistosome life cycle includes both developmental and constitutive patterns . Total RNA from indicated life-stages was obtained as described in Methods and utilized for real time quantitative PCR analysis to determine SmVAL1-13 transcript abundance. For each SmVAL transcript, a bar graph is displayed indicating relative abundance (compared to SmAT1) across the S. mansoni lifecycle. On the x -axis, each specific life-stage cDNA being tested is indicated. The y -axis represents the ratio of SmVAL gene expression relative to that of SmAT1 (reference gene). Data are presented as mean ratios (+/- standard deviation) from technical duplicates.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

    SmVAL gene clusters exist throughout the genome with SmVAL2, SmVAL8 and SmVAL12 genetically linked to chromosome 6 and W . A) Genomic regions containing two or more SmVAL genes were identified from interrogation of the current Schisto GeneDB v4 assembly. Schisto GeneDB v4 scaffold ID for each region is shown. SmVAL genes are shown as labelled boxes with the direction of transcription indicated by a triangle at the stop codon. The genomic sequence between SmVAL genes is represented with a dashed line and the length in base pairs shown below. B) Genomic linkage of SmVAL2, 8 and 12 was established by PCR amplification of gene specific regions from BAC clone Sm1-41J19. SmAT1 (alpha tubulin, M80214) was not contained on the BAC clone and was only amplified from adult worm cDNA. C) FISH analysis indicates positive signal (arrowheads) for Sm1-41J19 on chromosome 6 and W (W in inset). Bar indicates 10 μm.
    Figure Legend Snippet: SmVAL gene clusters exist throughout the genome with SmVAL2, SmVAL8 and SmVAL12 genetically linked to chromosome 6 and W . A) Genomic regions containing two or more SmVAL genes were identified from interrogation of the current Schisto GeneDB v4 assembly. Schisto GeneDB v4 scaffold ID for each region is shown. SmVAL genes are shown as labelled boxes with the direction of transcription indicated by a triangle at the stop codon. The genomic sequence between SmVAL genes is represented with a dashed line and the length in base pairs shown below. B) Genomic linkage of SmVAL2, 8 and 12 was established by PCR amplification of gene specific regions from BAC clone Sm1-41J19. SmAT1 (alpha tubulin, M80214) was not contained on the BAC clone and was only amplified from adult worm cDNA. C) FISH analysis indicates positive signal (arrowheads) for Sm1-41J19 on chromosome 6 and W (W in inset). Bar indicates 10 μm.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, BAC Assay, Fluorescence In Situ Hybridization

    26) Product Images from "XBP-1 specifically promotes IgM synthesis and secretion, but is dispensable for degradation of glycoproteins in primary B cells"

    Article Title: XBP-1 specifically promotes IgM synthesis and secretion, but is dispensable for degradation of glycoproteins in primary B cells

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20050575

    High levels of biosynthesis and secretion of IgM require XBP-1. (A) 10 6 live cells, as determined by trypan blue exclusion, were pulse-labeled with [ 35 S]methionine for 30 min. Cells were lysed in 1% SDS, and lysate was diluted to 0.07% SDS with NP-40 lysis mix followed by immunoprecipitation with anti-μ antibodies and analysis by SDS-PAGE (10%). (B) Autoradiograms were quantified by phosphoimager. Empty bars, WT B cells; black bars, XBP1 −/− B cells. (C) Cells stimulated for 3 d with LPS or CpG were pulse-labeled with [ 35 S]methionine for 30 min and chased for up to 4 h. Cells were lysed in 1% SDS; lysate was diluted to 0.07% SDS with NP-40 lysis mix followed by immunoprecipitation with anti-μ antibodies. At each time point, IgM also was recovered from the media by immunoprecipitated with anti-μ antibodies. Immunoprecipitates were analyzed by SDS-PAGE (10%). Each lane represents material from 10 6 live cells. (D) Autoradiograms were quantified by phosphoimager. Secreted μ chains were expressed as percentage from μ chains recovered after 4-h chase. (E) RNA was extracted from day 3 LPS-treated WT or XBP-1 −/− B cells and analyzed by Northern blotting. Quantification indicates a 2.6-fold reduction in μ mRNA levels in XBP-1 −/− cells. (F) Semi-quantitative RT-PCR analysis of cDNA prepared from RNA of day 3 LPS-treated WT or XBP-1 −/− B cells. Threefold dilution series of the cDNA were used as input material for the PCR with primers specific for μ chains or GAPDH as a reference. The analysis indicates a less than threefold reduction in μ mRNA levels in XBP-1 −/− cells.
    Figure Legend Snippet: High levels of biosynthesis and secretion of IgM require XBP-1. (A) 10 6 live cells, as determined by trypan blue exclusion, were pulse-labeled with [ 35 S]methionine for 30 min. Cells were lysed in 1% SDS, and lysate was diluted to 0.07% SDS with NP-40 lysis mix followed by immunoprecipitation with anti-μ antibodies and analysis by SDS-PAGE (10%). (B) Autoradiograms were quantified by phosphoimager. Empty bars, WT B cells; black bars, XBP1 −/− B cells. (C) Cells stimulated for 3 d with LPS or CpG were pulse-labeled with [ 35 S]methionine for 30 min and chased for up to 4 h. Cells were lysed in 1% SDS; lysate was diluted to 0.07% SDS with NP-40 lysis mix followed by immunoprecipitation with anti-μ antibodies. At each time point, IgM also was recovered from the media by immunoprecipitated with anti-μ antibodies. Immunoprecipitates were analyzed by SDS-PAGE (10%). Each lane represents material from 10 6 live cells. (D) Autoradiograms were quantified by phosphoimager. Secreted μ chains were expressed as percentage from μ chains recovered after 4-h chase. (E) RNA was extracted from day 3 LPS-treated WT or XBP-1 −/− B cells and analyzed by Northern blotting. Quantification indicates a 2.6-fold reduction in μ mRNA levels in XBP-1 −/− cells. (F) Semi-quantitative RT-PCR analysis of cDNA prepared from RNA of day 3 LPS-treated WT or XBP-1 −/− B cells. Threefold dilution series of the cDNA were used as input material for the PCR with primers specific for μ chains or GAPDH as a reference. The analysis indicates a less than threefold reduction in μ mRNA levels in XBP-1 −/− cells.

    Techniques Used: Labeling, Lysis, Immunoprecipitation, SDS Page, Northern Blot, Quantitative RT-PCR, Polymerase Chain Reaction

    XBP-1 controls plasmablasts' cell size. (A) WT or XBP-1 −/− B cells were purified from splenocytes by magnetic depletion with anti-CD43. Cells were plated at 10 6 cells/ml and stimulated with CpG. Flow cytometry analysis was performed every 24 h, and live cells were gated based on their forward and side scattering. Cells were replated at 10 6 cells/ml density and were analyzed the next day. Line graphs of the gated cells at the forward scatter channel (FSC) are shown in the right column. Gray, WT cells; white, XBP-1 −/− cells. The percentage of dead cells is indicated. (B) Cells were stimulated with CpG for 4 d. RNA was extracted at the indicated times, and splicing of XBP-1 mRNA was analyzed by RT-PCR. Tunicamycin treatment (1μg/ml, 4 h) of naive B cells was used as a positive control.
    Figure Legend Snippet: XBP-1 controls plasmablasts' cell size. (A) WT or XBP-1 −/− B cells were purified from splenocytes by magnetic depletion with anti-CD43. Cells were plated at 10 6 cells/ml and stimulated with CpG. Flow cytometry analysis was performed every 24 h, and live cells were gated based on their forward and side scattering. Cells were replated at 10 6 cells/ml density and were analyzed the next day. Line graphs of the gated cells at the forward scatter channel (FSC) are shown in the right column. Gray, WT cells; white, XBP-1 −/− cells. The percentage of dead cells is indicated. (B) Cells were stimulated with CpG for 4 d. RNA was extracted at the indicated times, and splicing of XBP-1 mRNA was analyzed by RT-PCR. Tunicamycin treatment (1μg/ml, 4 h) of naive B cells was used as a positive control.

    Techniques Used: Purification, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Positive Control

    27) Product Images from "DARPP-32 expression arises after a phase of dysplasia in oesophageal squamous cell carcinoma"

    Article Title: DARPP-32 expression arises after a phase of dysplasia in oesophageal squamous cell carcinoma

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601899

    RT–PCR expression analyses. ( A ) A gel imaging for DARPP-32, t-DARPP and β -actin expression at 30 cycles by RT–PCR. Seven oesophageal squamous cell lines, and positive controls (pCEP4-DARPP-32, pCEP4-t-DARPP) are shown. ( B ) DARPP protein expression. Seven oesophageal squamous cell lines and TE8 cells transfected with pCEP4-DARPP and pCEP4-t-DARPP were separated by SDS–PAGE and analysed for DARPP protein expression by Western blot with a COOH-terminal DARPP-32 antibody (Santa Cruz Biotechnology).
    Figure Legend Snippet: RT–PCR expression analyses. ( A ) A gel imaging for DARPP-32, t-DARPP and β -actin expression at 30 cycles by RT–PCR. Seven oesophageal squamous cell lines, and positive controls (pCEP4-DARPP-32, pCEP4-t-DARPP) are shown. ( B ) DARPP protein expression. Seven oesophageal squamous cell lines and TE8 cells transfected with pCEP4-DARPP and pCEP4-t-DARPP were separated by SDS–PAGE and analysed for DARPP protein expression by Western blot with a COOH-terminal DARPP-32 antibody (Santa Cruz Biotechnology).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Imaging, Transfection, SDS Page, Western Blot

    RT–PCR expression analyses. A gel imaging for DARPP-32, t-DARPP and β -actin expression at 30 cycles by RT–PCR. Seven different samples of normal oesophageal mucosa (N), tumour tissues (Ca) and positive control (pCEP4-DARPP-32, pCEP4-t-DARPP) are shown.
    Figure Legend Snippet: RT–PCR expression analyses. A gel imaging for DARPP-32, t-DARPP and β -actin expression at 30 cycles by RT–PCR. Seven different samples of normal oesophageal mucosa (N), tumour tissues (Ca) and positive control (pCEP4-DARPP-32, pCEP4-t-DARPP) are shown.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Imaging, Positive Control

    28) Product Images from "Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis"

    Article Title: Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.11.050

    The Irf8 Promoter Is Hypermethylated in Chronic Inflammation-Induced Colon Tumor (A) The mouse Irf8 promoter structure. The CpG islands are indicated by blue, transcription initiation site is indicated by +1. The numbers above the bar indicate the nucleotide location relative to the Irf8 transcription initiation site. Bottom: genomic DNA was extracted from the colon tissues of normal tumor-free mice (n = 3) and the colon tumor tissues of AOM-DSS-treated mice (n = 3) and modified with bisulfite. The modified genomic DNA was then amplified with bisulfite-modified DNA-specific primers to amplify a CpG island region, as indicated under the CpG island (–348 to +156). The amplified DNA fragments were cloned and sequenced. Each circle represents a CpG dinucleotide. Open circles indicate unmethylated CpG, and closed circles represent methylated CpG. (B) The bisulfite-modified genomic DNA as in (A) was also analyzed by methylation-specific (MS)-PCR. U, unmethylated; M, methylated. (C) Bisulfite-sequencing analysis of the CpG island region in the Irf8 promoter of colon epithelial CCD841 and colon carcinoma HCT116 cells. Each circle represents a CpG dinucleotide. Open circles indicate unmethylated CpG, and closed circles represent methylated CpG. (D) The Irf8 promoter DNA methylation datasets of normal human colon tissues and colorectal carcinoma tissues were extracted from TCGA database and compared. (E) MS-PCR analysis of the Irf8 promoter region in WT, DNMT1 −/− , DNMT3b −/− and DKO of HCT116 cells. U, unmethylated; M, methylated. (F) RNA was extracted from WT, DNMT1 −/− , DNMT3b −/− , and DKO of HCT116 cells and analyzed for IRF8 mRNA expression levels by semiquantitative RT-PCR (right top) and qPCR (left) using β-actin as an internal control. Bar, SD. Bottom right: the IRF8 protein level was analyzed by western blotting using IRF8-specific antibody. β-Actin was used as a normalization control.
    Figure Legend Snippet: The Irf8 Promoter Is Hypermethylated in Chronic Inflammation-Induced Colon Tumor (A) The mouse Irf8 promoter structure. The CpG islands are indicated by blue, transcription initiation site is indicated by +1. The numbers above the bar indicate the nucleotide location relative to the Irf8 transcription initiation site. Bottom: genomic DNA was extracted from the colon tissues of normal tumor-free mice (n = 3) and the colon tumor tissues of AOM-DSS-treated mice (n = 3) and modified with bisulfite. The modified genomic DNA was then amplified with bisulfite-modified DNA-specific primers to amplify a CpG island region, as indicated under the CpG island (–348 to +156). The amplified DNA fragments were cloned and sequenced. Each circle represents a CpG dinucleotide. Open circles indicate unmethylated CpG, and closed circles represent methylated CpG. (B) The bisulfite-modified genomic DNA as in (A) was also analyzed by methylation-specific (MS)-PCR. U, unmethylated; M, methylated. (C) Bisulfite-sequencing analysis of the CpG island region in the Irf8 promoter of colon epithelial CCD841 and colon carcinoma HCT116 cells. Each circle represents a CpG dinucleotide. Open circles indicate unmethylated CpG, and closed circles represent methylated CpG. (D) The Irf8 promoter DNA methylation datasets of normal human colon tissues and colorectal carcinoma tissues were extracted from TCGA database and compared. (E) MS-PCR analysis of the Irf8 promoter region in WT, DNMT1 −/− , DNMT3b −/− and DKO of HCT116 cells. U, unmethylated; M, methylated. (F) RNA was extracted from WT, DNMT1 −/− , DNMT3b −/− , and DKO of HCT116 cells and analyzed for IRF8 mRNA expression levels by semiquantitative RT-PCR (right top) and qPCR (left) using β-actin as an internal control. Bar, SD. Bottom right: the IRF8 protein level was analyzed by western blotting using IRF8-specific antibody. β-Actin was used as a normalization control.

    Techniques Used: Mouse Assay, Modification, Amplification, Clone Assay, Methylation, Mass Spectrometry, Polymerase Chain Reaction, Methylation Sequencing, DNA Methylation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot

    IL-10 Induces the Activation of STAT3 that Binds to the dnmt1 and dnmt3b Promoters in Colon Epithelial and Carcinoma Cells (A) WT C57BL/6 mice were treated with the 2% DSS-water cycle, as described in STAR Methods . Colon tissues were collected from mice at the indicated time points and analyzed by western blotting for STAT1 and STAT3 protein levels. β-Actin was used as a normalization control. (B) CCD841 and HT29 cells were treated with recombinant IL-10 (100 ng/mL) for 2 hr and analyzed for the indicated proteins by western blotting. (C) Top: structure of the Dnmt1 promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt1 transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using immunoglobulin G (IgG) control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt1 promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD. (D) Top: structure of the Dnmt3b promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt3b transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using IgG control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt3b promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD. (E and F) The human DNMT1 (E) and DNMT3b (F) promoter DNA fragments were amplified by PCR from the two indicated regions (top: P1 and P2 for DNMT1 , and P3 and P4 for DNMT3b ) and cloned to the pGL3 vector. pGL3 vectors containing the P1, P2, P3, or P4 DNA fragments were transiently transfected to CCD841 and HT29 cells, respectively, overnight. Cells were either untreated (control) or treated with IL-10 (100 ng/mL) for 4 hr. Cells were lysated and analyzed for luciferase activity. Bar, SD.
    Figure Legend Snippet: IL-10 Induces the Activation of STAT3 that Binds to the dnmt1 and dnmt3b Promoters in Colon Epithelial and Carcinoma Cells (A) WT C57BL/6 mice were treated with the 2% DSS-water cycle, as described in STAR Methods . Colon tissues were collected from mice at the indicated time points and analyzed by western blotting for STAT1 and STAT3 protein levels. β-Actin was used as a normalization control. (B) CCD841 and HT29 cells were treated with recombinant IL-10 (100 ng/mL) for 2 hr and analyzed for the indicated proteins by western blotting. (C) Top: structure of the Dnmt1 promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt1 transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using immunoglobulin G (IgG) control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt1 promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD. (D) Top: structure of the Dnmt3b promoter region. The number below the bar indicates nucleotide locations relative to the Dnmt3b transcription initiation site. The ChIP PCR primer regions are indicated under the bar. Bottom: CCD841 and HT29 cells were stimulated with recombinant IL-10 protein (100 ng/mL) for 16 hr, then analyzed by ChIP using IgG control antibody and pSTAT3-specific antibody, respectively, followed by qPCR analysis with Dnmt3b promoter DNA-specific PCR primers, as shown at top. Input DNA was used as a normalization control. The input of each ChIP primer set was arbitrarily set at 1, and the pSTAT3 was normalized to the input DNA level. Column, mean; bar, SD. (E and F) The human DNMT1 (E) and DNMT3b (F) promoter DNA fragments were amplified by PCR from the two indicated regions (top: P1 and P2 for DNMT1 , and P3 and P4 for DNMT3b ) and cloned to the pGL3 vector. pGL3 vectors containing the P1, P2, P3, or P4 DNA fragments were transiently transfected to CCD841 and HT29 cells, respectively, overnight. Cells were either untreated (control) or treated with IL-10 (100 ng/mL) for 4 hr. Cells were lysated and analyzed for luciferase activity. Bar, SD.

    Techniques Used: Activation Assay, Mouse Assay, Western Blot, Recombinant, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Transfection, Luciferase, Activity Assay

    IL-10 Upregulates DNMT1 and DNMT3b Expression in Colon Epithelial and Carcinoma Cells (A) Colonic epithelial CCD841 and colon carcinoma HT29 cells were treated with recombinant IL-10 at the indicated doses for 24 hr and analyzed for DNMT1 and DNMT3b mRNA expression levels by semiquantitative RT-PCR (top) and qPCR (bottom) using β-actin as an internal control. Bar, SD. (B) CCD841 and HT29 cells were treated with recombinant IL-10 (100 ng/mL) for 24 hr and analyzed by western blotting analysis of DNMT1, DNMT3b (left), and IRF8 (right) protein levels. (C) WT (n = 4) and IL-10 KO (n = 5) mice were treated with the DSS-water cycle, as described in STAR Methods for 28 days. Colon tissues were collected and analyzed by western blotting for DNMT1 and DNMT3b protein levels. (D) WT (n = 3) and IL-10 KO (n = 3) mice were treated with the 2% DSS-water cycle, as described in STAR Methods . Colon tissues were collected at day 28 and analyzed by qPCR for the IRF8 mRNA level with β-actin as an internal control. Bar, SD. (E) The pGL3 vector containing the human IRF8 promoter was treated with methylase in vitro and transfected to CCD841 cells overnight. Cells were lysated and analyzed for luciferase activity, as described in STAR Methods .
    Figure Legend Snippet: IL-10 Upregulates DNMT1 and DNMT3b Expression in Colon Epithelial and Carcinoma Cells (A) Colonic epithelial CCD841 and colon carcinoma HT29 cells were treated with recombinant IL-10 at the indicated doses for 24 hr and analyzed for DNMT1 and DNMT3b mRNA expression levels by semiquantitative RT-PCR (top) and qPCR (bottom) using β-actin as an internal control. Bar, SD. (B) CCD841 and HT29 cells were treated with recombinant IL-10 (100 ng/mL) for 24 hr and analyzed by western blotting analysis of DNMT1, DNMT3b (left), and IRF8 (right) protein levels. (C) WT (n = 4) and IL-10 KO (n = 5) mice were treated with the DSS-water cycle, as described in STAR Methods for 28 days. Colon tissues were collected and analyzed by western blotting for DNMT1 and DNMT3b protein levels. (D) WT (n = 3) and IL-10 KO (n = 3) mice were treated with the 2% DSS-water cycle, as described in STAR Methods . Colon tissues were collected at day 28 and analyzed by qPCR for the IRF8 mRNA level with β-actin as an internal control. Bar, SD. (E) The pGL3 vector containing the human IRF8 promoter was treated with methylase in vitro and transfected to CCD841 cells overnight. Cells were lysated and analyzed for luciferase activity, as described in STAR Methods .

    Techniques Used: Expressing, Recombinant, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Mouse Assay, Plasmid Preparation, In Vitro, Transfection, Luciferase, Activity Assay

    29) Product Images from "Alternative splicing and nonsense-mediated decay regulate telomerase reverse transcriptase (TERT) expression during virus-induced lymphomagenesis in vivo"

    Article Title: Alternative splicing and nonsense-mediated decay regulate telomerase reverse transcriptase (TERT) expression during virus-induced lymphomagenesis in vivo

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-10-571

    Alternative transcripts of chTERT identified in three avian cell line . At the top, we show a schematic diagram of telomerase protein, showing major conserved protein motifs, including reverse transcriptase domains 1 and 2 and the A, B, C, D and E motifs, and of the chTERT gene, with its 16 exons shown as gray boxes. The PCR primers are shown as arrows below the diagram. Each alternative transcript is shown on the right, with the splicing event depicted as a clear gray box for insertion of the exon cassette; dark lines indicate deletion, black boxes indicate intron retention and white boxes indicate the deletion of part of an exon. Positions of premature stop codons (PTCs) are indicated by black triangles. On the left, we show the name of the spliced transcripts, the presence or absence of a PTC with its position relative to the 3' exon-exon junction and represented as a function of cell line. The name of the transcript is indicated on the left and is coded as follow: iXec for insertion of exon cassette X, dXf for full deletion of exon X, dXp for partial deletion of exon X, iXp for retention of part of intron X and iXf for insertion of full intron X.
    Figure Legend Snippet: Alternative transcripts of chTERT identified in three avian cell line . At the top, we show a schematic diagram of telomerase protein, showing major conserved protein motifs, including reverse transcriptase domains 1 and 2 and the A, B, C, D and E motifs, and of the chTERT gene, with its 16 exons shown as gray boxes. The PCR primers are shown as arrows below the diagram. Each alternative transcript is shown on the right, with the splicing event depicted as a clear gray box for insertion of the exon cassette; dark lines indicate deletion, black boxes indicate intron retention and white boxes indicate the deletion of part of an exon. Positions of premature stop codons (PTCs) are indicated by black triangles. On the left, we show the name of the spliced transcripts, the presence or absence of a PTC with its position relative to the 3' exon-exon junction and represented as a function of cell line. The name of the transcript is indicated on the left and is coded as follow: iXec for insertion of exon cassette X, dXf for full deletion of exon X, dXp for partial deletion of exon X, iXp for retention of part of intron X and iXf for insertion of full intron X.

    Techniques Used: Polymerase Chain Reaction

    Downregulation of the i10ce variant by NMD . (A) Upf1 depletion increases levels of the chTERT splice variant i10ce. Levels of i10ce are expressed relative to total chTERT levels. Peak areas of i10ec were obtained by capillary electrophoresis analysis of the PCR products targeting exon 10, generated from cDNA from 1 well of P6 transfected with 25 pmol or 50 pmol of siUPF-1. The histogram shows the mean and standard deviation obtained from 4 biological analyses. All values are expressed as a fold difference with respect to
    Figure Legend Snippet: Downregulation of the i10ce variant by NMD . (A) Upf1 depletion increases levels of the chTERT splice variant i10ce. Levels of i10ce are expressed relative to total chTERT levels. Peak areas of i10ec were obtained by capillary electrophoresis analysis of the PCR products targeting exon 10, generated from cDNA from 1 well of P6 transfected with 25 pmol or 50 pmol of siUPF-1. The histogram shows the mean and standard deviation obtained from 4 biological analyses. All values are expressed as a fold difference with respect to "no silencing" (NS), for which the value was fixed at 1. (B) siUpf1 knockdown of Upf1. The histogram shows Upf1 mRNA levels, calculated by RT-PCR analysis. Upf1 levels in each sample were normalized with respect to GAPDH levels for the corresponding sample. The values obtained for "no silencing" (NS) LMH cells were set at 100%.

    Techniques Used: Variant Assay, Electrophoresis, Polymerase Chain Reaction, Generated, Transfection, Standard Deviation, Reverse Transcription Polymerase Chain Reaction

    Regulation of chTERT splicing involving variants 5 and 10 during lymphomagenesis in vivo . Proportions of constitutively spliced chTERT transcript (in blue) and alternatively spliced variants d10f+i10ec (in yellow) and the d5f variant (in green), as shown in the panels on the right and left, respectively. The proportions correspond to the peak area obtained by capillary electrophoresis analysis of PCR targeting exon 5 (A) or 10 (B), performed on cDNA extracted from sorted CD4 + T cells sampled from GaHV-2-infected chickens at 5 different time points after infection, as indicated on the x-axis. The curve shows telomerase activity, which was obtained by summing the peak areas corresponding to the elongation products (right y-axis) (C) Proportions of d10f (green) and i10ec (orange) variants, as indicated above the graph and corresponding to 100% of alternative splice variants10 at each time point after infection, as shown in (B).
    Figure Legend Snippet: Regulation of chTERT splicing involving variants 5 and 10 during lymphomagenesis in vivo . Proportions of constitutively spliced chTERT transcript (in blue) and alternatively spliced variants d10f+i10ec (in yellow) and the d5f variant (in green), as shown in the panels on the right and left, respectively. The proportions correspond to the peak area obtained by capillary electrophoresis analysis of PCR targeting exon 5 (A) or 10 (B), performed on cDNA extracted from sorted CD4 + T cells sampled from GaHV-2-infected chickens at 5 different time points after infection, as indicated on the x-axis. The curve shows telomerase activity, which was obtained by summing the peak areas corresponding to the elongation products (right y-axis) (C) Proportions of d10f (green) and i10ec (orange) variants, as indicated above the graph and corresponding to 100% of alternative splice variants10 at each time point after infection, as shown in (B).

    Techniques Used: In Vivo, Variant Assay, Electrophoresis, Polymerase Chain Reaction, Infection, Activity Assay

    30) Product Images from "A tyrosine sulfation–dependent HLA-I modification identifies memory B cells and plasma cells"

    Article Title: A tyrosine sulfation–dependent HLA-I modification identifies memory B cells and plasma cells

    Journal: Science Advances

    doi: 10.1126/sciadv.aar7653

    VLRB N8 recognizes a tyrosine sulfation–dependent antigen on HLA-I. ( A ) KMS-11 cells were cultured in the presence of the indicated concentrations of NaClO 3 for 48 hours followed by flow cytometric assessment of VLRB N8 and HLA-I reactivity. A representative experiment is depicted in the top panel, and VLRB N8/HLA-I ratios from five independent experiments are shown in the bottom bar diagram, depicted as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s post test ( n = 5). ( B ) Inhibition of VLRB N8 recognition of HLA-I on BJAB cells following PMA and ionomycin stimulation. Cells were stimulated for 1 hour with PMA and ionomycin, and VLRB N8 and HLA-I binding were assessed following a 36-hour culture with the indicated concentrations of NaClO 3 . Means ± SD of VLRB N8 signals normalized to HLA-I are shown. Statistical significance was determined using two-way ANOVA test with Dunnett’s post test ( n = 4). ( C ) shRNA-mediated down-regulation of transduced BJAB cells was verified by qRT-PCR. Transcript levels of TPST1 (left) and TPST2 (right) of the indicated cell populations are depicted as means ± SD ( n = 3). Statistical significance was determined using a Student’s t test. ( D ) shRNA-transduced BJAB cells were stimulated with anti-Ig (20 μg/ml), followed by the assessment of VLRB N8 and anti–HLA-I recognition. Numbers indicate the mean fold induction of HLA-I normalized VLRB N8. Statistical significance for induced VLRB N8 binding was determined using a one-way ANOVA test with Tukey’s post test ( n = 9). ( E ) Tyrosine sulfation of HLA-I following antigen receptor engagement. A representative autoradiogram (left) of anti–HLA-I immunoprecipitates of unstimulated and stimulated BJAB cells and the quantitation (right) of six independent experiments are shown. 35 SO 4 incorporation is shown with arbitrary units (AU). Statistical significance was determined using paired Student’s t test ( n = 6). ( F ) TPST1 and TPST2 transcript analysis of tonsillar B cell populations. Means ± SD of qRT-PCR of TPST1 or TPST2 normalized to HPRT from five independent tonsil specimens are shown. Statistically significant differences of P
    Figure Legend Snippet: VLRB N8 recognizes a tyrosine sulfation–dependent antigen on HLA-I. ( A ) KMS-11 cells were cultured in the presence of the indicated concentrations of NaClO 3 for 48 hours followed by flow cytometric assessment of VLRB N8 and HLA-I reactivity. A representative experiment is depicted in the top panel, and VLRB N8/HLA-I ratios from five independent experiments are shown in the bottom bar diagram, depicted as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s post test ( n = 5). ( B ) Inhibition of VLRB N8 recognition of HLA-I on BJAB cells following PMA and ionomycin stimulation. Cells were stimulated for 1 hour with PMA and ionomycin, and VLRB N8 and HLA-I binding were assessed following a 36-hour culture with the indicated concentrations of NaClO 3 . Means ± SD of VLRB N8 signals normalized to HLA-I are shown. Statistical significance was determined using two-way ANOVA test with Dunnett’s post test ( n = 4). ( C ) shRNA-mediated down-regulation of transduced BJAB cells was verified by qRT-PCR. Transcript levels of TPST1 (left) and TPST2 (right) of the indicated cell populations are depicted as means ± SD ( n = 3). Statistical significance was determined using a Student’s t test. ( D ) shRNA-transduced BJAB cells were stimulated with anti-Ig (20 μg/ml), followed by the assessment of VLRB N8 and anti–HLA-I recognition. Numbers indicate the mean fold induction of HLA-I normalized VLRB N8. Statistical significance for induced VLRB N8 binding was determined using a one-way ANOVA test with Tukey’s post test ( n = 9). ( E ) Tyrosine sulfation of HLA-I following antigen receptor engagement. A representative autoradiogram (left) of anti–HLA-I immunoprecipitates of unstimulated and stimulated BJAB cells and the quantitation (right) of six independent experiments are shown. 35 SO 4 incorporation is shown with arbitrary units (AU). Statistical significance was determined using paired Student’s t test ( n = 6). ( F ) TPST1 and TPST2 transcript analysis of tonsillar B cell populations. Means ± SD of qRT-PCR of TPST1 or TPST2 normalized to HPRT from five independent tonsil specimens are shown. Statistically significant differences of P

    Techniques Used: Cell Culture, Flow Cytometry, Inhibition, Binding Assay, shRNA, Quantitative RT-PCR, Quantitation Assay

    31) Product Images from "P Body-Associated Protein Mov10 Inhibits HIV-1 Replication at Multiple Stages ▿P Body-Associated Protein Mov10 Inhibits HIV-1 Replication at Multiple Stages ▿ †"

    Article Title: P Body-Associated Protein Mov10 Inhibits HIV-1 Replication at Multiple Stages ▿P Body-Associated Protein Mov10 Inhibits HIV-1 Replication at Multiple Stages ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.00585-10

    Real-time PCR analysis of the effects of A3G and Mov10 on HIV-1 reverse transcription. (A) The effects of F-A3G (2 μg DNA) and F-Mov10 (4 μg) on pHDV-EGFP infectivity were determined by flow cytometry. HDV-EGFP produced in the absence
    Figure Legend Snippet: Real-time PCR analysis of the effects of A3G and Mov10 on HIV-1 reverse transcription. (A) The effects of F-A3G (2 μg DNA) and F-Mov10 (4 μg) on pHDV-EGFP infectivity were determined by flow cytometry. HDV-EGFP produced in the absence

    Techniques Used: Real-time Polymerase Chain Reaction, Infection, Flow Cytometry, Cytometry, Produced

    32) Product Images from "Expression of t-DARPP Mediates Trastuzumab Resistance in Breast Cancer Cells"

    Article Title: Expression of t-DARPP Mediates Trastuzumab Resistance in Breast Cancer Cells

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-08-0121

    Transcriptional up-regulation of t-DARPP in trastuzumab-resistant cell lines is not associated with gene amplification or promoter hypomethylation. Gene-specific primers for DNA- and mRNA-specific sequences of t-DARPP, ERBB2, β-actin , and HPRT1 were used for PCR and real-time PCR in BT-474, HR-5, and HR-6 cells; the results were normalized to β-actin and HPRT1. A , t-DARPP mRNA expression levels in HR-5 and HR-6 cells were 25- to 100-fold higher compared with BT-474 cells (*, P ≤ 0.001). In contrast, t-DARPP gene amplification levels were similar in all cells. B , ERBB2 mRNA expression and gene amplification levels remained unchanged in all cells. C , DNA bisulfite treatment and pyrosequencing analysis of DNA methylation. DNA from BT-474, HR-5, and HR-6 cells was extracted and modified by bisulfite treatment as described in Materials and Methods. A CpG island from -1,438 to -830 of t-DARPP was amplified with PCR using specific primers. The PCR products were then subjected to pyrosequencing analysis to determine DNA methylation of 10 CpG sites within -1,161 to -1,109 region of t-DARPP. Comparable levels of DNA methylation were detected in all three cell lines, indicating that DNA methylation of t-DARPP CpG island does not regulate its expression in these cell lines.
    Figure Legend Snippet: Transcriptional up-regulation of t-DARPP in trastuzumab-resistant cell lines is not associated with gene amplification or promoter hypomethylation. Gene-specific primers for DNA- and mRNA-specific sequences of t-DARPP, ERBB2, β-actin , and HPRT1 were used for PCR and real-time PCR in BT-474, HR-5, and HR-6 cells; the results were normalized to β-actin and HPRT1. A , t-DARPP mRNA expression levels in HR-5 and HR-6 cells were 25- to 100-fold higher compared with BT-474 cells (*, P ≤ 0.001). In contrast, t-DARPP gene amplification levels were similar in all cells. B , ERBB2 mRNA expression and gene amplification levels remained unchanged in all cells. C , DNA bisulfite treatment and pyrosequencing analysis of DNA methylation. DNA from BT-474, HR-5, and HR-6 cells was extracted and modified by bisulfite treatment as described in Materials and Methods. A CpG island from -1,438 to -830 of t-DARPP was amplified with PCR using specific primers. The PCR products were then subjected to pyrosequencing analysis to determine DNA methylation of 10 CpG sites within -1,161 to -1,109 region of t-DARPP. Comparable levels of DNA methylation were detected in all three cell lines, indicating that DNA methylation of t-DARPP CpG island does not regulate its expression in these cell lines.

    Techniques Used: Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, DNA Methylation Assay, Modification

    33) Product Images from "Podocyte-specific chemokine (C-C motif) receptor 2 overexpression mediates diabetic renal injury in mice"

    Article Title: Podocyte-specific chemokine (C-C motif) receptor 2 overexpression mediates diabetic renal injury in mice

    Journal: Kidney international

    doi: 10.1016/j.kint.2016.09.042

    Effect of podocin2.5-CCR2 expression on kidney fibronectin and type-1 collagen mRNA expression in diabetic mice Quantitative RT-PCR was performed on whole mouse kidney total RNA after 9 weeks following diabetes. Fibronectin ( A ), and type-1 collagen ( B ) mRNA expression were normalized with GAPDH mRNA. Open bar, control groups; black-filled bar, diabetic groups. Results are means ± SEM. * p
    Figure Legend Snippet: Effect of podocin2.5-CCR2 expression on kidney fibronectin and type-1 collagen mRNA expression in diabetic mice Quantitative RT-PCR was performed on whole mouse kidney total RNA after 9 weeks following diabetes. Fibronectin ( A ), and type-1 collagen ( B ) mRNA expression were normalized with GAPDH mRNA. Open bar, control groups; black-filled bar, diabetic groups. Results are means ± SEM. * p

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

    Effect of podocin2.5-CCR2 expression on kidney inflammatory cytokines in diabetic mice Quantitative RT-PCR was performed on whole mouse kidney total RNA after 9 weeks following diabetes. TNF-α ( A ), and NOS2 ( B ) mRNA expression was normalized with GAPDH mRNA. MSD multi-spot assay system was performed to measure TNF-α (C), and IL-2 (D) protein expression in kidney tissues. Open bar, control groups; black-filled bar, diabetic groups. Results are means ± SEM. * p
    Figure Legend Snippet: Effect of podocin2.5-CCR2 expression on kidney inflammatory cytokines in diabetic mice Quantitative RT-PCR was performed on whole mouse kidney total RNA after 9 weeks following diabetes. TNF-α ( A ), and NOS2 ( B ) mRNA expression was normalized with GAPDH mRNA. MSD multi-spot assay system was performed to measure TNF-α (C), and IL-2 (D) protein expression in kidney tissues. Open bar, control groups; black-filled bar, diabetic groups. Results are means ± SEM. * p

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Spot Test

    34) Product Images from "Domain analysis reveals striking functional differences between the regulatory subunits of phosphatidylinositol 3-kinase (PI3K), p85α and p85β"

    Article Title: Domain analysis reveals striking functional differences between the regulatory subunits of phosphatidylinositol 3-kinase (PI3K), p85α and p85β

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19866

    Expression of the isolated iSH2 domains enhances oncogenic and signaling activity of PI3K A. Representative focus assays of p85α, p85β, iSH2α and iSH2β. Both iSH2 domains induce a thickening of the cell sheet that is indicative of oncogenic transformation. The effect is not as pronounced as that induced by p85β, but is evident in comparison with the non-transforming p85α. B. Western blots documenting enhanced PI3K signaling with pAKT S473 and pS6. Expression of the iSH2α domain with the HA tag was not clearly demonstrable in standard Western blots and was therefore documented with qRTPCR C. The positive controls in this figure represent standard PCR from the DNA constructs; the HA iSH2α and iSH2β bands were generated by qRTPCR.
    Figure Legend Snippet: Expression of the isolated iSH2 domains enhances oncogenic and signaling activity of PI3K A. Representative focus assays of p85α, p85β, iSH2α and iSH2β. Both iSH2 domains induce a thickening of the cell sheet that is indicative of oncogenic transformation. The effect is not as pronounced as that induced by p85β, but is evident in comparison with the non-transforming p85α. B. Western blots documenting enhanced PI3K signaling with pAKT S473 and pS6. Expression of the iSH2α domain with the HA tag was not clearly demonstrable in standard Western blots and was therefore documented with qRTPCR C. The positive controls in this figure represent standard PCR from the DNA constructs; the HA iSH2α and iSH2β bands were generated by qRTPCR.

    Techniques Used: Expressing, Isolation, Activity Assay, Transformation Assay, Western Blot, Polymerase Chain Reaction, Construct, Generated

    35) Product Images from "Characterization of Nrf1b, a Novel Isoform of the Nuclear Factor-Erythroid-2 Related Transcription Factor-1 That Activates Antioxidant Response Element-Regulated Genes"

    Article Title: Characterization of Nrf1b, a Novel Isoform of the Nuclear Factor-Erythroid-2 Related Transcription Factor-1 That Activates Antioxidant Response Element-Regulated Genes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0048404

    Nrf1b expression is widely distributed. Nrf1a and Nrf1b mRNA expression patterns were analyzed by RT-PCR in various cell lines ( A ) and mouse tissues ( B ) . Nrf1a and Nrf1b cDNA was amplified by PCR for 30 cycles and 18S was amplified for 20 cycles. Histograms show relative Nrf1a and Nrf1b expression normalized against 18S. ( C ) . Western blot of different mouse tissues probed with Nrf1 antibody. HEK293 cells transfected with pEF1-Nrf1a (lane 1), and pEF1-Nrf1b (lane 2) were used as controls for detection of the Nrf1a and Nrf1b isoforms by the Nrf1 antibody. Beta-actin was used as a loading control.
    Figure Legend Snippet: Nrf1b expression is widely distributed. Nrf1a and Nrf1b mRNA expression patterns were analyzed by RT-PCR in various cell lines ( A ) and mouse tissues ( B ) . Nrf1a and Nrf1b cDNA was amplified by PCR for 30 cycles and 18S was amplified for 20 cycles. Histograms show relative Nrf1a and Nrf1b expression normalized against 18S. ( C ) . Western blot of different mouse tissues probed with Nrf1 antibody. HEK293 cells transfected with pEF1-Nrf1a (lane 1), and pEF1-Nrf1b (lane 2) were used as controls for detection of the Nrf1a and Nrf1b isoforms by the Nrf1 antibody. Beta-actin was used as a loading control.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Western Blot, Transfection

    36) Product Images from "Sensitivity to TOP2 Targeting Chemotherapeutics Is Regulated by Oct1 and FILIP1L"

    Article Title: Sensitivity to TOP2 Targeting Chemotherapeutics Is Regulated by Oct1 and FILIP1L

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042921

    A functional shRNA screen for regulators of doxorubicin induced apoptosis. (A) Outline of the doxorubicin induced apoptosis bypass screen using U2OS cells. Pools of shRNA were transfected into retroviral packaging cell lines, and retrovirus transduced into U2OS cells followed by puromycin selection. Transduced U2OS cells were treated with 225 ng/ml Doxorubicin for 5 days, which led to apoptotic death of approximately 99.8% of the library infected cells. We harvested cells that survived treatment, isolated genomic DNA, PCR amplified the region containing shRNA sequences, shotgun cloned and sequenced. A total of approximately 1500 inserts were sequenced. (B) Twelve genes identified by this screen are listed. Full gene names and the number of times identified are also listed.
    Figure Legend Snippet: A functional shRNA screen for regulators of doxorubicin induced apoptosis. (A) Outline of the doxorubicin induced apoptosis bypass screen using U2OS cells. Pools of shRNA were transfected into retroviral packaging cell lines, and retrovirus transduced into U2OS cells followed by puromycin selection. Transduced U2OS cells were treated with 225 ng/ml Doxorubicin for 5 days, which led to apoptotic death of approximately 99.8% of the library infected cells. We harvested cells that survived treatment, isolated genomic DNA, PCR amplified the region containing shRNA sequences, shotgun cloned and sequenced. A total of approximately 1500 inserts were sequenced. (B) Twelve genes identified by this screen are listed. Full gene names and the number of times identified are also listed.

    Techniques Used: Functional Assay, shRNA, Transfection, Selection, Infection, Isolation, Polymerase Chain Reaction, Amplification, Clone Assay

    37) Product Images from "Rod Photoreceptor Ribbon Synapses in DBA/2J Mice Show Progressive Age-Related Structural Changes"

    Article Title: Rod Photoreceptor Ribbon Synapses in DBA/2J Mice Show Progressive Age-Related Structural Changes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044645

    C1qA and C1qC gene expression is upregulated in photoreceptor cells of aging DBA/2J mice. (A) Agarose gels showing PCR fragments that represent the gene expression of C1qA, C1qB and C1qC in the retinae of 2 and 6 months old DBA/2J and C57BL/6 control mice. (B) The expression of C1qA, C1qB and C1qC in photoreceptor cells of 2, 6, and 10 months old DBA/2J mice and age-matched C57BL/6 control mice was compared with PCR. For each age, cDNA obtained from three animals was subjected to triplicate PCR amplifications (n = 9). Statistically significant differences are indicated by asterisks (* p
    Figure Legend Snippet: C1qA and C1qC gene expression is upregulated in photoreceptor cells of aging DBA/2J mice. (A) Agarose gels showing PCR fragments that represent the gene expression of C1qA, C1qB and C1qC in the retinae of 2 and 6 months old DBA/2J and C57BL/6 control mice. (B) The expression of C1qA, C1qB and C1qC in photoreceptor cells of 2, 6, and 10 months old DBA/2J mice and age-matched C57BL/6 control mice was compared with PCR. For each age, cDNA obtained from three animals was subjected to triplicate PCR amplifications (n = 9). Statistically significant differences are indicated by asterisks (* p

    Techniques Used: Expressing, Mouse Assay, Polymerase Chain Reaction

    38) Product Images from "A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites"

    Article Title: A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites

    Journal: eLife

    doi: 10.7554/eLife.03582

    Genotype analysis of the generated PfΔ slarp and PfΔ b9 Δ slarp parasites . ( A ) Long range PCR analysis of genomic DNA from WT, Pf Δ slarp and Pf Δ b9 Δ slarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B ) and PCR products are also digested with restriction enzymes x ( Xma I) and kx ( Kpn I/ Xcm I) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δ slarp-a , 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δ slarp-b , 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δ b9-b , 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). ( B ) Southern analysis of restricted genomic DNA from WT, PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 asexual parasites. DNA was digested with restriction enzyme (E: Taq I) and probed with the 5′ slarp targeting region (P: 5′ slarp -T; see A ) on the left side of the slarp Southern or probed with the 3′ slarp targeting region (P: 3′ slarp -T; see A ) on the right side of the slarp panel. For analysis of the b9 , integration DNA was digested with restriction enzymes (E: Rca I) and probed with the 5′ b9 targeting region (P: 5′ b9 -T; see A ) on the right panel. The expected fragment sizes are indicated in panel ( A ). ( C ) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B ) and generated the expected 130 bp fragment. DOI: http://dx.doi.org/10.7554/eLife.03582.009
    Figure Legend Snippet: Genotype analysis of the generated PfΔ slarp and PfΔ b9 Δ slarp parasites . ( A ) Long range PCR analysis of genomic DNA from WT, Pf Δ slarp and Pf Δ b9 Δ slarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B ) and PCR products are also digested with restriction enzymes x ( Xma I) and kx ( Kpn I/ Xcm I) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δ slarp-a , 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δ slarp-b , 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δ b9-b , 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). ( B ) Southern analysis of restricted genomic DNA from WT, PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 asexual parasites. DNA was digested with restriction enzyme (E: Taq I) and probed with the 5′ slarp targeting region (P: 5′ slarp -T; see A ) on the left side of the slarp Southern or probed with the 3′ slarp targeting region (P: 3′ slarp -T; see A ) on the right side of the slarp panel. For analysis of the b9 , integration DNA was digested with restriction enzymes (E: Rca I) and probed with the 5′ b9 targeting region (P: 5′ b9 -T; see A ) on the right panel. The expected fragment sizes are indicated in panel ( A ). ( C ) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B ) and generated the expected 130 bp fragment. DOI: http://dx.doi.org/10.7554/eLife.03582.009

    Techniques Used: Generated, Polymerase Chain Reaction, Marker, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Amplification, Purification, Positive Control

    Generation and genotype analyses of P. berghei mutant PbΔ slarp- a . ( A ) Generation of mutant PbΔ slarp -a. For PbΔ slarp -a, the DNA-construct pL1740 was generated containing the positive/negative selectable marker cassette hdhfr / yfcy . This construct was subsequently used to generate the mutant PbΔ slarp -a (1839cl3) in the Pb GFP-Luc con reference line. See Supplementary file 2A for the sequence of the primers. ( B ) Diagnostic PCR and Southern analysis of Pulse Field Gel (PFG)-separated chromosomes of mutant Δ slarp -a confirming correct disruption of the slarp -locus. See Supplementary file 2A for the sequence of the primers used for the selectable marker gene (SM); 5′-integration event (5′); 3′-integration event (3′); and the slarp ORF. Mutant PbΔ slarp -a has been generated in the reference P. berghei ANKA line Pb GFP-Luc con which has a gfp-luciferase gene integrated into the silent 230p locus (PBANKA_030600) on chromosome 3. For Southern analysis, PFG-separated chromosomes were hybridized using a 3′UTR pbdhfr probe that recognizes the construct integrated into P. berghei slarp locus on chromosome 9, the endogenous locus of dhfr/ts on chromosome 7, and the gfp-luciferase gene integrated into chromosome 3. In addition, the chromosomes were hybridized with the hdhfr probe recognizing the integrated construct into the slarp locus on chromosome 9. ( C ) Real time in vivo imaging of Δslarp luciferase-expressing liver-stage parasites in C57BL/6 mice at 24, 35, and 45 hr post-infection. C57BL/6 mice were IV injected with either 5 × 10 4 Pb -GFPLuc con sporozoites (n = 5), resulting in a full liver infection (upper panel: representative image of WT infected mice), or with 5 × 10 5 Pb Δslarp-a sporozoites (n = 5) (lower panel: representative image of Pb Δslarp-luc infected mice). DOI: http://dx.doi.org/10.7554/eLife.03582.006
    Figure Legend Snippet: Generation and genotype analyses of P. berghei mutant PbΔ slarp- a . ( A ) Generation of mutant PbΔ slarp -a. For PbΔ slarp -a, the DNA-construct pL1740 was generated containing the positive/negative selectable marker cassette hdhfr / yfcy . This construct was subsequently used to generate the mutant PbΔ slarp -a (1839cl3) in the Pb GFP-Luc con reference line. See Supplementary file 2A for the sequence of the primers. ( B ) Diagnostic PCR and Southern analysis of Pulse Field Gel (PFG)-separated chromosomes of mutant Δ slarp -a confirming correct disruption of the slarp -locus. See Supplementary file 2A for the sequence of the primers used for the selectable marker gene (SM); 5′-integration event (5′); 3′-integration event (3′); and the slarp ORF. Mutant PbΔ slarp -a has been generated in the reference P. berghei ANKA line Pb GFP-Luc con which has a gfp-luciferase gene integrated into the silent 230p locus (PBANKA_030600) on chromosome 3. For Southern analysis, PFG-separated chromosomes were hybridized using a 3′UTR pbdhfr probe that recognizes the construct integrated into P. berghei slarp locus on chromosome 9, the endogenous locus of dhfr/ts on chromosome 7, and the gfp-luciferase gene integrated into chromosome 3. In addition, the chromosomes were hybridized with the hdhfr probe recognizing the integrated construct into the slarp locus on chromosome 9. ( C ) Real time in vivo imaging of Δslarp luciferase-expressing liver-stage parasites in C57BL/6 mice at 24, 35, and 45 hr post-infection. C57BL/6 mice were IV injected with either 5 × 10 4 Pb -GFPLuc con sporozoites (n = 5), resulting in a full liver infection (upper panel: representative image of WT infected mice), or with 5 × 10 5 Pb Δslarp-a sporozoites (n = 5) (lower panel: representative image of Pb Δslarp-luc infected mice). DOI: http://dx.doi.org/10.7554/eLife.03582.006

    Techniques Used: Mutagenesis, Construct, Generated, Marker, Sequencing, Diagnostic Assay, Polymerase Chain Reaction, Luciferase, In Vivo Imaging, Expressing, Mouse Assay, Infection, Injection

    39) Product Images from "Zinc finger nuclease-mediated CCR5 knockout hematopoietic stem cell transplantation controls HIV-1 in vivo"

    Article Title: Zinc finger nuclease-mediated CCR5 knockout hematopoietic stem cell transplantation controls HIV-1 in vivo

    Journal: Nature biotechnology

    doi: 10.1038/nbt.1663

    ZFN activity produces heterogeneous mutations in CCR5. Sequence analysis was performed on 60 cloned human CCR5 alleles, PCR amplified from intraepithelial cells from the large intestine of an HIV-infected mouse previously transplanted with ZFN-treated HSC, and at 12 weeks post-infection. The number of nucleotides deleted or inserted at the ZFN target site in each clone is indicated on the right of each sequence, together with the number of times the sequence was found. Dashes (-) indicate deleted bases compared to the wildtype sequence, uppercase letters are point mutations, underlined upper case letters are inserted bases. Some specific mutations of CCR5 occurred more frequently, in particular a 5bp duplication at the ZFN target site that was identified 13 times (bottom sequence). No mutations in CCR5 were observed in a similar analysis performed on control samples from a mouse receiving non-modified HSC (data not shown).
    Figure Legend Snippet: ZFN activity produces heterogeneous mutations in CCR5. Sequence analysis was performed on 60 cloned human CCR5 alleles, PCR amplified from intraepithelial cells from the large intestine of an HIV-infected mouse previously transplanted with ZFN-treated HSC, and at 12 weeks post-infection. The number of nucleotides deleted or inserted at the ZFN target site in each clone is indicated on the right of each sequence, together with the number of times the sequence was found. Dashes (-) indicate deleted bases compared to the wildtype sequence, uppercase letters are point mutations, underlined upper case letters are inserted bases. Some specific mutations of CCR5 occurred more frequently, in particular a 5bp duplication at the ZFN target site that was identified 13 times (bottom sequence). No mutations in CCR5 were observed in a similar analysis performed on control samples from a mouse receiving non-modified HSC (data not shown).

    Techniques Used: Activity Assay, Sequencing, Clone Assay, Polymerase Chain Reaction, Amplification, Infection, Modification

    ZFN-mediated disruption of CCR5 gene in HSC. ( a ) Representative gel showing extent of CCR5 disruption in CD34+ HSC, 24 hours after cells were nucleofected with ZFN-expressing plasmids (ZFN) or mock nucleofected (mock). Neg. is untreated HSC. CCR5 disruption was measured by PCR amplification across the ZFN target site, followed by Cel I nuclease digestion and quantitation of products by PAGE. ( b ) Graph showing mean +/− SD percentage of human CD45+ cells in peripheral blood of mice at 8 weeks post-transplantation with either untreated, mock nucleofected, or ZFN nucleofected HSC, (n=5 each group). ( c ) FACS profiles of human cells from various organs of one representative mouse transplanted with ZFN-treated HSC. Cells were gated on FSC/SSC to remove debris. Staining for human CD45, a pan leukocyte marker, was used to reveal the level of human cell engraftment in each organ. CD45+ gated populations were further analyzed for subsets, as indicated: CD19 (B cells) in bone marrow, CD14 (monocytes/macrophages) in lung, CD4 and CD8 (T cells) in thymus and spleen, and CD3 (T cells) in the small intestine (lamina propria). The CD45+ population from the small intestine was further analyzed for CD4 and CCR5 expression. Peripheral blood cells from CD45+ and lymphoid gates were analyzed for CD4 and CD8 expression. The percentage of cells in each indicated area is shown. No staining was observed with isotype-matched control antibodies ( Supplementary Fig. 1 online), or in animals receiving no human graft (data not shown).
    Figure Legend Snippet: ZFN-mediated disruption of CCR5 gene in HSC. ( a ) Representative gel showing extent of CCR5 disruption in CD34+ HSC, 24 hours after cells were nucleofected with ZFN-expressing plasmids (ZFN) or mock nucleofected (mock). Neg. is untreated HSC. CCR5 disruption was measured by PCR amplification across the ZFN target site, followed by Cel I nuclease digestion and quantitation of products by PAGE. ( b ) Graph showing mean +/− SD percentage of human CD45+ cells in peripheral blood of mice at 8 weeks post-transplantation with either untreated, mock nucleofected, or ZFN nucleofected HSC, (n=5 each group). ( c ) FACS profiles of human cells from various organs of one representative mouse transplanted with ZFN-treated HSC. Cells were gated on FSC/SSC to remove debris. Staining for human CD45, a pan leukocyte marker, was used to reveal the level of human cell engraftment in each organ. CD45+ gated populations were further analyzed for subsets, as indicated: CD19 (B cells) in bone marrow, CD14 (monocytes/macrophages) in lung, CD4 and CD8 (T cells) in thymus and spleen, and CD3 (T cells) in the small intestine (lamina propria). The CD45+ population from the small intestine was further analyzed for CD4 and CCR5 expression. Peripheral blood cells from CD45+ and lymphoid gates were analyzed for CD4 and CD8 expression. The percentage of cells in each indicated area is shown. No staining was observed with isotype-matched control antibodies ( Supplementary Fig. 1 online), or in animals receiving no human graft (data not shown).

    Techniques Used: Expressing, Polymerase Chain Reaction, Amplification, Quantitation Assay, Polyacrylamide Gel Electrophoresis, Mouse Assay, Transplantation Assay, FACS, Staining, Marker

    40) Product Images from "Opposing roles of mitochondrial and nuclear PARP1 in the regulation of mitochondrial and nuclear DNA integrity: implications for the regulation of mitochondrial function"

    Article Title: Opposing roles of mitochondrial and nuclear PARP1 in the regulation of mitochondrial and nuclear DNA integrity: implications for the regulation of mitochondrial function

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1089

    Effect of PARP1 depletion and PARP1 inhibition on the integrity and repair of the mitochondrial DNA. ( A ) Integrity of the mitochondrial and nuclear DNA in baseline conditions of A549 shCTR and A549 shPARP1 cells was determined by LA-PCR. The protein level of PARP1 was analyzed by western blot and is shown in the inset. ( B ) Sensitivity of the mitochondrial DNA to increasing levels of oxidative stress, generated by increasing concentrations of glucose oxidase (GOx) measured by LA-PCR after 1 h of GOx treatment. ( C ) Repair of the mitochondrial DNA integrity in a control (shCTR) and PARP1-depleted (shPARP1) A549 cells. ( D ) Repair of the mitochondrial DNA integrity in parental A549 cells transiently transfected with scrambled (siCTR) or PARP1-specific (siPARP1) siRNA. ( E ) Repair of the mitochondrial DNA integrity in A549 cells preincubated with the PARP inhibitor PJ34 (10 μM) for 30 min. The graphs represent means±SD calculated based on at least two independent experiments run in triplicates (* P
    Figure Legend Snippet: Effect of PARP1 depletion and PARP1 inhibition on the integrity and repair of the mitochondrial DNA. ( A ) Integrity of the mitochondrial and nuclear DNA in baseline conditions of A549 shCTR and A549 shPARP1 cells was determined by LA-PCR. The protein level of PARP1 was analyzed by western blot and is shown in the inset. ( B ) Sensitivity of the mitochondrial DNA to increasing levels of oxidative stress, generated by increasing concentrations of glucose oxidase (GOx) measured by LA-PCR after 1 h of GOx treatment. ( C ) Repair of the mitochondrial DNA integrity in a control (shCTR) and PARP1-depleted (shPARP1) A549 cells. ( D ) Repair of the mitochondrial DNA integrity in parental A549 cells transiently transfected with scrambled (siCTR) or PARP1-specific (siPARP1) siRNA. ( E ) Repair of the mitochondrial DNA integrity in A549 cells preincubated with the PARP inhibitor PJ34 (10 μM) for 30 min. The graphs represent means±SD calculated based on at least two independent experiments run in triplicates (* P

    Techniques Used: Inhibition, Polymerase Chain Reaction, Western Blot, Generated, Transfection

    41) Product Images from "PGC-1α overexpression suppresses blood pressure elevation in DOCA-salt hypertensive mice"

    Article Title: PGC-1α overexpression suppresses blood pressure elevation in DOCA-salt hypertensive mice

    Journal: Bioscience Reports

    doi: 10.1042/BSR20150076

    Reduced PGC-1 α is found in the aortas of DOCA-salt hypertensive mice ( A and B ) PGC-1α expression in aortas of mice treated with DOCA-salt over a 3-week period was determined by western blot ( A ) and quantitative PCR ( B ). ** P
    Figure Legend Snippet: Reduced PGC-1 α is found in the aortas of DOCA-salt hypertensive mice ( A and B ) PGC-1α expression in aortas of mice treated with DOCA-salt over a 3-week period was determined by western blot ( A ) and quantitative PCR ( B ). ** P

    Techniques Used: Pyrolysis Gas Chromatography, Mouse Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    42) Product Images from "Using Recombineering to Generate Point Mutations: galK-Based Positive-Negative Selection Method"

    Article Title: Using Recombineering to Generate Point Mutations: galK-Based Positive-Negative Selection Method

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-61779-564-0_10

    Schematic representation of galK based recombineering to generate point mutations A . Generation of targeting vector by PCR to insert the galK cassette using primers P1 and P2. 3´ end of each P1 and P2 anneal to the 5´ and 3´ ends of the galK cassette, respectively. The 5´ ends of the primers have 70 bases of homology to the target site. B . The targeting vector to replace the galK cassette with desired mutation (marked in bold in the box) is generated by PCR using two 100-mer oligonucleotides, P3 and P4. The two oligonucleotides have complementary 3´ ends where they anneal together and amplify the homology arms (HA) to generate the targeting vector. C . The two steps depicting the recombineering procedure to generate point mutation. The base to be replaced in the wild-type BAC and the mutated base in mutant BAC is marked in bold letter in the box. P5 and P6 mark the two PCR primers located outside the homology arms that can be used for screening the correctly targeted clones in both steps of recombineering.
    Figure Legend Snippet: Schematic representation of galK based recombineering to generate point mutations A . Generation of targeting vector by PCR to insert the galK cassette using primers P1 and P2. 3´ end of each P1 and P2 anneal to the 5´ and 3´ ends of the galK cassette, respectively. The 5´ ends of the primers have 70 bases of homology to the target site. B . The targeting vector to replace the galK cassette with desired mutation (marked in bold in the box) is generated by PCR using two 100-mer oligonucleotides, P3 and P4. The two oligonucleotides have complementary 3´ ends where they anneal together and amplify the homology arms (HA) to generate the targeting vector. C . The two steps depicting the recombineering procedure to generate point mutation. The base to be replaced in the wild-type BAC and the mutated base in mutant BAC is marked in bold letter in the box. P5 and P6 mark the two PCR primers located outside the homology arms that can be used for screening the correctly targeted clones in both steps of recombineering.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Mutagenesis, Generated, BAC Assay, Clone Assay

    43) Product Images from "Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci"

    Article Title: Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci

    Journal: eLife

    doi: 10.7554/eLife.13926

    Evidence of retrotransposition capability for selected L1HS-Ta copies. ( a ) Evidence of retrotransposition competence for the top 20 most expressed L1 copies across all cell lines analyzed. Cellular assays refer to retrotransposition cellular assays of plasmid-borne L1 instances, whose expression is driven by either the native L1 5’ UTR alone ( Brouha et al., 2003 ) or supplemented by a strong CMV promoter ([ Beck et al., 2010 ] and Figure 5b ). These assays measure L1 intrinsic biochemical activity, independently of their actual expression in their genomic context. Three-prime transduction refers to the existence of progeny copies containing a 3' transduction, which can be traced back to the original locus and reflect a retrotransposition event. ( b ) Retrotransposition assay in cultured cells for MCF7 L1 copy EXP_ID_0447 ( NEDD4 locus). A full length transcribed L1HS-Ta copy present in the genome of MCF7 cells was subcloned by PCR in an expression vector containing a reporter gene to measure retrotransposition activity and generated four independent clones (pVan610-1 to -4). In transfected HeLa cells, de novo retrotransposition events of engineered L1 copies lead to the introduction of a functional genomic copy of the neomycin phosphotransferase gene, which expression confers resistance to G418. Resistant foci were stained and counted to monitor retrotransposition activity compared to the positive (pJM101/L1.3, wild type L1HS-Ta) and negative (pJM105/L1.3, mutant L1HS-Ta) control conditions. The value of G418 resistant colonies obtained with the positive control was set to 100%. A picture of a representative well with stained colonies is displayed for illustrative purposes under each bar of the graph. The average value of three biological replicates is displayed with error bars corresponding to the standard deviation among the three biological replicates. ( c ) Detection of 3' transductions in ATLAS-seq data. This in silico screen identifies L1HS-Ta copy (progeny element) with ATLAS-seq clusters containing reads with non-aligning subsequences (soft-clipped), which uniquely map downstream and adjacent to another full length L1HS locus (progenitor element). The panel shows a genome browser view of such a 3' transduction, originating from a full length L1HS-Ta in the TTC28 gene (22q12.1). The soft-clipped region of the reads is shown in color (base code: T, red; A, green; C, blue; G; orange). As expected, the transduced region is flanked by 2 poly(A) tails (poly(T) here since it is located on the reverse genomic strand). DOI: http://dx.doi.org/10.7554/eLife.13926.014
    Figure Legend Snippet: Evidence of retrotransposition capability for selected L1HS-Ta copies. ( a ) Evidence of retrotransposition competence for the top 20 most expressed L1 copies across all cell lines analyzed. Cellular assays refer to retrotransposition cellular assays of plasmid-borne L1 instances, whose expression is driven by either the native L1 5’ UTR alone ( Brouha et al., 2003 ) or supplemented by a strong CMV promoter ([ Beck et al., 2010 ] and Figure 5b ). These assays measure L1 intrinsic biochemical activity, independently of their actual expression in their genomic context. Three-prime transduction refers to the existence of progeny copies containing a 3' transduction, which can be traced back to the original locus and reflect a retrotransposition event. ( b ) Retrotransposition assay in cultured cells for MCF7 L1 copy EXP_ID_0447 ( NEDD4 locus). A full length transcribed L1HS-Ta copy present in the genome of MCF7 cells was subcloned by PCR in an expression vector containing a reporter gene to measure retrotransposition activity and generated four independent clones (pVan610-1 to -4). In transfected HeLa cells, de novo retrotransposition events of engineered L1 copies lead to the introduction of a functional genomic copy of the neomycin phosphotransferase gene, which expression confers resistance to G418. Resistant foci were stained and counted to monitor retrotransposition activity compared to the positive (pJM101/L1.3, wild type L1HS-Ta) and negative (pJM105/L1.3, mutant L1HS-Ta) control conditions. The value of G418 resistant colonies obtained with the positive control was set to 100%. A picture of a representative well with stained colonies is displayed for illustrative purposes under each bar of the graph. The average value of three biological replicates is displayed with error bars corresponding to the standard deviation among the three biological replicates. ( c ) Detection of 3' transductions in ATLAS-seq data. This in silico screen identifies L1HS-Ta copy (progeny element) with ATLAS-seq clusters containing reads with non-aligning subsequences (soft-clipped), which uniquely map downstream and adjacent to another full length L1HS locus (progenitor element). The panel shows a genome browser view of such a 3' transduction, originating from a full length L1HS-Ta in the TTC28 gene (22q12.1). The soft-clipped region of the reads is shown in color (base code: T, red; A, green; C, blue; G; orange). As expected, the transduced region is flanked by 2 poly(A) tails (poly(T) here since it is located on the reverse genomic strand). DOI: http://dx.doi.org/10.7554/eLife.13926.014

    Techniques Used: Plasmid Preparation, Expressing, Activity Assay, Transduction, Cell Culture, Polymerase Chain Reaction, Generated, Clone Assay, Transfection, Functional Assay, Staining, Mutagenesis, Positive Control, Standard Deviation, In Silico

    RT-PCR validation of individual L1 expression across several cell lines. PCR primers are anchored in the L1 internal sequence and in the flanking genomic region, respectively. Each RT-PCR included a control reaction without RT (-) to exclude possible genomic DNA contamination. Top, RT-PCR reactions. Bottom, PCR on genomic DNA using the same primers, showing polymorphic L1 copies among the various cell lines, and validating PCR conditions. RT, reverse transcriptase. DOI: http://dx.doi.org/10.7554/eLife.13926.012
    Figure Legend Snippet: RT-PCR validation of individual L1 expression across several cell lines. PCR primers are anchored in the L1 internal sequence and in the flanking genomic region, respectively. Each RT-PCR included a control reaction without RT (-) to exclude possible genomic DNA contamination. Top, RT-PCR reactions. Bottom, PCR on genomic DNA using the same primers, showing polymorphic L1 copies among the various cell lines, and validating PCR conditions. RT, reverse transcriptase. DOI: http://dx.doi.org/10.7554/eLife.13926.012

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Sequencing

    44) Product Images from "Expression of the p66Shc protein adaptor is regulated by the activator of transcription STAT4 in normal and chronic lymphocytic leukemia B cells"

    Article Title: Expression of the p66Shc protein adaptor is regulated by the activator of transcription STAT4 in normal and chronic lymphocytic leukemia B cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10977

    Impaired STAT4 expression in primary CLL B cells A. qRT-PCR analysis of p66shc , STAT4 , IL10 , IL1β and IFNγ mRNA in purified peripheral B cells from either healthy donors (Ctr, n=9) or CLL patients with mutated (M; n=15) or unmutated (U; n=15) IGHV . B. Representative immunoblot analysis of STAT4 in 2 healthy donors, 2 M-CLL and 2 U-CLL patients. The numbers below the representative blot refer to the quantification of STAT4 immunoreactive band in B-cell lysates from 10 M-CLL, 9 U-CLL and 6 healthy controls, of which at least 1 was included in each gel as reference. C. qRT-PCR analysis of STAT4 mRNA in CLL B cells nucleofected with either empty vector (CLL-Ctr) or an expression construct encoding p66Shc (CLL-p66). The relative abundance of STAT4 transcript was determined on triplicate samples from each patient using the ΔΔCt method and is expressed as the normalized fold expression (mean±SD; empty vector controls taken as 1 for all CLL samples). All samples (n=6) were checked for reconstitution of p66shc expression by qRT-PCR (data not shown). A representative immunoblot of STAT4 and p66Shc is shown on the right. Filters were reprobed for actin as loading control. The histogram shows the quantification of STAT4 in B-cell lysates from 3 reconstituted CLL samples. *** P ≤ 0.001; ** P ≤0.01; and * P ≤0.05.
    Figure Legend Snippet: Impaired STAT4 expression in primary CLL B cells A. qRT-PCR analysis of p66shc , STAT4 , IL10 , IL1β and IFNγ mRNA in purified peripheral B cells from either healthy donors (Ctr, n=9) or CLL patients with mutated (M; n=15) or unmutated (U; n=15) IGHV . B. Representative immunoblot analysis of STAT4 in 2 healthy donors, 2 M-CLL and 2 U-CLL patients. The numbers below the representative blot refer to the quantification of STAT4 immunoreactive band in B-cell lysates from 10 M-CLL, 9 U-CLL and 6 healthy controls, of which at least 1 was included in each gel as reference. C. qRT-PCR analysis of STAT4 mRNA in CLL B cells nucleofected with either empty vector (CLL-Ctr) or an expression construct encoding p66Shc (CLL-p66). The relative abundance of STAT4 transcript was determined on triplicate samples from each patient using the ΔΔCt method and is expressed as the normalized fold expression (mean±SD; empty vector controls taken as 1 for all CLL samples). All samples (n=6) were checked for reconstitution of p66shc expression by qRT-PCR (data not shown). A representative immunoblot of STAT4 and p66Shc is shown on the right. Filters were reprobed for actin as loading control. The histogram shows the quantification of STAT4 in B-cell lysates from 3 reconstituted CLL samples. *** P ≤ 0.001; ** P ≤0.01; and * P ≤0.05.

    Techniques Used: Expressing, Quantitative RT-PCR, Purification, Plasmid Preparation, Construct

    STAT4 regulates p66shc expression A. In silico promoter analysis of the human p66shc promoter highlighting several putative STAT4 binding sites (grey shaded regions). SP1 binding sites identified in the analysis and known to be present in the p66shc promoter are shown in bold. The transcription start site (ATG) is also shown (underlined, italic). B. Top , schematic representation of the p66shc region taken into consideration for the ChIP experiments. Black arrows indicate the position of the primers used, dark squares the position of the STAT4 binding sites. The p66shc TSS is located in exon 2. Bottom , Nuclear extracts of EBV-B cells were subjected to ChIP assay with an antibody specific for STAT4. Precipitated DNA was amplified by qRT-PCR using primers described above. Unspecific IgG was used as control. Data are presented as percentage of input DNA (mean±SD; n=6). C. Scheme of the in vitro binding experiments. EBV-B nuclear extracts were used to pull down STAT4 bound to the biotinylated p66shc promoter, and bound STAT4 was detected by immunoblot. Lanes 1, 3 and 4 correspond to the beads bound to the promoter region containing one, three or all four STAT4 binding sites, respectively. Streptavidin beads incubated with nuclear extract were used as negative control (lane 0). Pull-down experiment using mutants of the putative STAT4 binding sites (M) showed no STAT4 immunoreactive band compared to the control (WT). (n=3). D-F. p66shc promoter activation assays in EBV-B cells. (D) Cells were co-transfected with reporter plasmids carrying p66shc promoter sequences containing none (pGL4p66Shc-460/+71), one (pGL4p66Shc-800/+71), or all (pGL4p66Shc-1300/+71) STAT4 binding sites, or empty vector (Ctr). A co-transfected Renilla plasmid was used as normalization control. (E) EBV-B cells were co-transfected with the pGL4p66Shc-1300/+71 luciferase reporter, the Renilla transfection control and a plasmid encoding STAT4. (F) EBV-B cells were co-transfected with plasmids containing the mutated STAT4-binding sequences or the corresponding WT sequences, and the Renilla transfection control. *** P ≤0.001; ** P ≤0.01, and * P ≤0.05 (n=3)
    Figure Legend Snippet: STAT4 regulates p66shc expression A. In silico promoter analysis of the human p66shc promoter highlighting several putative STAT4 binding sites (grey shaded regions). SP1 binding sites identified in the analysis and known to be present in the p66shc promoter are shown in bold. The transcription start site (ATG) is also shown (underlined, italic). B. Top , schematic representation of the p66shc region taken into consideration for the ChIP experiments. Black arrows indicate the position of the primers used, dark squares the position of the STAT4 binding sites. The p66shc TSS is located in exon 2. Bottom , Nuclear extracts of EBV-B cells were subjected to ChIP assay with an antibody specific for STAT4. Precipitated DNA was amplified by qRT-PCR using primers described above. Unspecific IgG was used as control. Data are presented as percentage of input DNA (mean±SD; n=6). C. Scheme of the in vitro binding experiments. EBV-B nuclear extracts were used to pull down STAT4 bound to the biotinylated p66shc promoter, and bound STAT4 was detected by immunoblot. Lanes 1, 3 and 4 correspond to the beads bound to the promoter region containing one, three or all four STAT4 binding sites, respectively. Streptavidin beads incubated with nuclear extract were used as negative control (lane 0). Pull-down experiment using mutants of the putative STAT4 binding sites (M) showed no STAT4 immunoreactive band compared to the control (WT). (n=3). D-F. p66shc promoter activation assays in EBV-B cells. (D) Cells were co-transfected with reporter plasmids carrying p66shc promoter sequences containing none (pGL4p66Shc-460/+71), one (pGL4p66Shc-800/+71), or all (pGL4p66Shc-1300/+71) STAT4 binding sites, or empty vector (Ctr). A co-transfected Renilla plasmid was used as normalization control. (E) EBV-B cells were co-transfected with the pGL4p66Shc-1300/+71 luciferase reporter, the Renilla transfection control and a plasmid encoding STAT4. (F) EBV-B cells were co-transfected with plasmids containing the mutated STAT4-binding sequences or the corresponding WT sequences, and the Renilla transfection control. *** P ≤0.001; ** P ≤0.01, and * P ≤0.05 (n=3)

    Techniques Used: Expressing, In Silico, Binding Assay, Chromatin Immunoprecipitation, Amplification, Quantitative RT-PCR, In Vitro, Incubation, Negative Control, Activation Assay, Transfection, Plasmid Preparation, Luciferase

    STAT4 modulates the levels of p66Shc in B cells A. qRT-PCR analysis of STAT4 and p66shc mRNA in EBV-B cells transiently transfected with either empty vector (Ctr) or an expression construct encoding STAT4 (STAT4). B. Immunoblot analysis of p46Shc, p52Shc and p66Shc 48 h after transfection with the STAT4-encoding construct in EBV-B cells. The histogram shows the quantification of the p66Shc immunoreactive band (n≥3). C. EBV-B cells were transfected with esiRNA targeting STAT4 and the levels of p66shc and STAT4 were measured by qRT-PCR. The relative abundance of the gene transcripts was determined on triplicate samples from at least 3 independent experiments using the ΔΔCt method and is expressed as the normalized fold expression (mean±SD). D. Immunoblot analysis of p46Shc, p52Shc and p66Shc 48 h after transfection of EBV-B cells with esiRNA targeting STAT4 (n≥3). Filters were reprobed for actin as loading control. The histogram shows the quantification of the p66Shc immunoreactive band. ** P ≤0.01; and * P ≤0.05.
    Figure Legend Snippet: STAT4 modulates the levels of p66Shc in B cells A. qRT-PCR analysis of STAT4 and p66shc mRNA in EBV-B cells transiently transfected with either empty vector (Ctr) or an expression construct encoding STAT4 (STAT4). B. Immunoblot analysis of p46Shc, p52Shc and p66Shc 48 h after transfection with the STAT4-encoding construct in EBV-B cells. The histogram shows the quantification of the p66Shc immunoreactive band (n≥3). C. EBV-B cells were transfected with esiRNA targeting STAT4 and the levels of p66shc and STAT4 were measured by qRT-PCR. The relative abundance of the gene transcripts was determined on triplicate samples from at least 3 independent experiments using the ΔΔCt method and is expressed as the normalized fold expression (mean±SD). D. Immunoblot analysis of p46Shc, p52Shc and p66Shc 48 h after transfection of EBV-B cells with esiRNA targeting STAT4 (n≥3). Filters were reprobed for actin as loading control. The histogram shows the quantification of the p66Shc immunoreactive band. ** P ≤0.01; and * P ≤0.05.

    Techniques Used: Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, Construct, esiRNA

    Activation of STAT4 promotes p66Shc transcription and B-cell apoptosis A. Nuclear extracts of EBV-B cells were subjected to ChIP assay with an antibody specific for p-STAT4. Precipitated DNA was amplified by qRT-PCR. Unspecific IgG was used as control. Data are presented as percentage of input DNA (mean±SD; n=6). B. Reporter gene assays on EBV-B cells co-transfected with the p66shc promoter reporter plasmid (pGL4p66Shc-460/+71) containing one STAT4 site and the control Renilla plasmid. Cells were stimulated or not with the IL-12 for 3 h prior to measuring luciferase activity (n > 3). C. qRT-PCR analysis of p66shc and STAT4 mRNA in EBV-B cells as above in presence or absence of 20 μM LSF (n≥3). D. Immunoblot analysis of STAT4, pSTAT4 and the three Shc isoforms in EBV-B cells stimulated or not with IL-12 in presence or absence of 20 μM LSF. Filters were reprobed for actin as loading control. The histograms show the quantification of STAT4, p-STAT4 and p66Shc, normalized to actin (n=3). E. Flow cytometric analysis of AnnexinV + /PI − EBV-B cells stimulated or not with IL-12 in presence or absence of 20 μM LSF followed by treatment with 500 ng/ml A23187. F. Flow cytometric analysis of AnnexinV + /PI − EBV-B cells transfected with siRNAs targeting p66shc or STAT4 , respectively, and stimulated with IL-12/A23187. Cells transfected with a scrambled siRNA were used as control. Data represent the mean±SD of the Annexin V + /PI − cells (n=3). *** P ≤ 0.001; ** P ≤0.01; and * P ≤0.05.
    Figure Legend Snippet: Activation of STAT4 promotes p66Shc transcription and B-cell apoptosis A. Nuclear extracts of EBV-B cells were subjected to ChIP assay with an antibody specific for p-STAT4. Precipitated DNA was amplified by qRT-PCR. Unspecific IgG was used as control. Data are presented as percentage of input DNA (mean±SD; n=6). B. Reporter gene assays on EBV-B cells co-transfected with the p66shc promoter reporter plasmid (pGL4p66Shc-460/+71) containing one STAT4 site and the control Renilla plasmid. Cells were stimulated or not with the IL-12 for 3 h prior to measuring luciferase activity (n > 3). C. qRT-PCR analysis of p66shc and STAT4 mRNA in EBV-B cells as above in presence or absence of 20 μM LSF (n≥3). D. Immunoblot analysis of STAT4, pSTAT4 and the three Shc isoforms in EBV-B cells stimulated or not with IL-12 in presence or absence of 20 μM LSF. Filters were reprobed for actin as loading control. The histograms show the quantification of STAT4, p-STAT4 and p66Shc, normalized to actin (n=3). E. Flow cytometric analysis of AnnexinV + /PI − EBV-B cells stimulated or not with IL-12 in presence or absence of 20 μM LSF followed by treatment with 500 ng/ml A23187. F. Flow cytometric analysis of AnnexinV + /PI − EBV-B cells transfected with siRNAs targeting p66shc or STAT4 , respectively, and stimulated with IL-12/A23187. Cells transfected with a scrambled siRNA were used as control. Data represent the mean±SD of the Annexin V + /PI − cells (n=3). *** P ≤ 0.001; ** P ≤0.01; and * P ≤0.05.

    Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Amplification, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Flow Cytometry

    p66Shc affects STAT4 protein stability A. qRT-PCR analysis of STAT4 and p66shc mRNA in EBV-B cells transfected with siRNA targeting p66shc . The relative abundance of the genes transcripts as described above and is expressed as the normalized fold expression (mean±SD, (n≥3). B. Immunoblot analysis of STAT4 and the three Shc isoforms in EBV-B cells transfected with a siRNA targeting p66shc . Filters were reprobed for actin as loading control. The histogram shows the quantification of the levels of STAT4, normalized to actin (n≥3). *** P ≤0.001; and ** P
    Figure Legend Snippet: p66Shc affects STAT4 protein stability A. qRT-PCR analysis of STAT4 and p66shc mRNA in EBV-B cells transfected with siRNA targeting p66shc . The relative abundance of the genes transcripts as described above and is expressed as the normalized fold expression (mean±SD, (n≥3). B. Immunoblot analysis of STAT4 and the three Shc isoforms in EBV-B cells transfected with a siRNA targeting p66shc . Filters were reprobed for actin as loading control. The histogram shows the quantification of the levels of STAT4, normalized to actin (n≥3). *** P ≤0.001; and ** P

    Techniques Used: Quantitative RT-PCR, Transfection, Expressing

    p66Shc alters the expression of several genes linked to the IL-12 pathway in B cells A, B, D. qRT-PCR analysis of IFNγ, IL1B, IL10, IFNGR1, IL18RAP, IL7R and STAT4 mRNA in MEC-1 cells stably transfected with a construct encoding p66Shc (MEC-p66) or empty vector (MEC-Ctr). The relative abundance of gene transcripts was determined on triplicate samples from ≥3 independent mRNA extractions using the ΔΔCt method and is expressed as normalized fold expression (mean±SD). C. Flow cytometric analysis of intracellular IFN-γ, IL-10 and IL-1β in MEC-Ctr and MEC-p66 cells treated for 24 h with a combination of PMA and A23187 in the presence of brefeldin A. Data are expressed as mean fluorescence intensity (MFI) ± SD (n≥3). *** P ≤0.001, ** P ≤0.01, * P ≤0.05.
    Figure Legend Snippet: p66Shc alters the expression of several genes linked to the IL-12 pathway in B cells A, B, D. qRT-PCR analysis of IFNγ, IL1B, IL10, IFNGR1, IL18RAP, IL7R and STAT4 mRNA in MEC-1 cells stably transfected with a construct encoding p66Shc (MEC-p66) or empty vector (MEC-Ctr). The relative abundance of gene transcripts was determined on triplicate samples from ≥3 independent mRNA extractions using the ΔΔCt method and is expressed as normalized fold expression (mean±SD). C. Flow cytometric analysis of intracellular IFN-γ, IL-10 and IL-1β in MEC-Ctr and MEC-p66 cells treated for 24 h with a combination of PMA and A23187 in the presence of brefeldin A. Data are expressed as mean fluorescence intensity (MFI) ± SD (n≥3). *** P ≤0.001, ** P ≤0.01, * P ≤0.05.

    Techniques Used: Expressing, Quantitative RT-PCR, Stable Transfection, Transfection, Construct, Plasmid Preparation, Flow Cytometry, Fluorescence

    45) Product Images from "Nestin overexpression precedes caspase-3 upregulation in rats exposed to controlled cortical impact traumatic brain injury"

    Article Title: Nestin overexpression precedes caspase-3 upregulation in rats exposed to controlled cortical impact traumatic brain injury

    Journal: Cell Medicine

    doi:

    QRT-PCR analyses of nestin and caspase-3 expression in TBI brains. QRT-PCR was conducted using the entire brain. (A) confirms RNA integrity under UV light by visualization of 28S- and 18S-rRNA bands on a denaturing gel containing ethidium bromide. (B)
    Figure Legend Snippet: QRT-PCR analyses of nestin and caspase-3 expression in TBI brains. QRT-PCR was conducted using the entire brain. (A) confirms RNA integrity under UV light by visualization of 28S- and 18S-rRNA bands on a denaturing gel containing ethidium bromide. (B)

    Techniques Used: Quantitative RT-PCR, Expressing

    46) Product Images from "The nuclear receptor constitutive active/androstane receptor (CAR) arrests DNA-damaged human hepatocellular carcinoma Huh7 cells at the G2/M phase"

    Article Title: The nuclear receptor constitutive active/androstane receptor (CAR) arrests DNA-damaged human hepatocellular carcinoma Huh7 cells at the G2/M phase

    Journal: Molecular carcinogenesis

    doi: 10.1002/mc.20783

    Transition of the cell cycle status and the candidate GADD45γ and Cyclin G2 mRNA expression after adriamycin treatment. (A) Huh7 cells were infected with adenovirus-β-Gal or adenovirus-mCAR at 10 MOI and following treated with TCPOBOP. Then, treated cells were collected after indicated time course of adriamycin treatment and the DNA content was analyzed by flow cytometry to determine the cell cycle distribution. The percentage of cells at the G0/G1 and at the G2/M phase in each group was summarized. This experiment was reproducibly repeated three times. (B) Treated Huh7 cells were cultured on duplicated dishes and were harvested after indicated time course of adriamycin treatment. Total RNAs were prepared from cells and were subjected to real-time PCR, and normalized to that of human B-actin mRNA. The relative levels of GADD45β and Cyclin G2 mRNA were shown relative to those of corresponding mRNAs in NI / DMSO / adriamycin 0 hr treatment sample set at one. The data are reported as mean ± SD, n=2. (C) Treated Huh7 cells were cultured on duplicated dishes and were harvested after indicated time course of adriamycin treatment. Total RNAs were prepared from cells and were subjected to real-time PCR, and normalized to that of human B-actin mRNA. The relative levels of GADD45α and GADD45β mRNA were shown relative to those of corresponding mRNAs in NI / DMSO / adriamycin 0 hr treatment sample set at one. The data are reported as mean ± SD, n=2.
    Figure Legend Snippet: Transition of the cell cycle status and the candidate GADD45γ and Cyclin G2 mRNA expression after adriamycin treatment. (A) Huh7 cells were infected with adenovirus-β-Gal or adenovirus-mCAR at 10 MOI and following treated with TCPOBOP. Then, treated cells were collected after indicated time course of adriamycin treatment and the DNA content was analyzed by flow cytometry to determine the cell cycle distribution. The percentage of cells at the G0/G1 and at the G2/M phase in each group was summarized. This experiment was reproducibly repeated three times. (B) Treated Huh7 cells were cultured on duplicated dishes and were harvested after indicated time course of adriamycin treatment. Total RNAs were prepared from cells and were subjected to real-time PCR, and normalized to that of human B-actin mRNA. The relative levels of GADD45β and Cyclin G2 mRNA were shown relative to those of corresponding mRNAs in NI / DMSO / adriamycin 0 hr treatment sample set at one. The data are reported as mean ± SD, n=2. (C) Treated Huh7 cells were cultured on duplicated dishes and were harvested after indicated time course of adriamycin treatment. Total RNAs were prepared from cells and were subjected to real-time PCR, and normalized to that of human B-actin mRNA. The relative levels of GADD45α and GADD45β mRNA were shown relative to those of corresponding mRNAs in NI / DMSO / adriamycin 0 hr treatment sample set at one. The data are reported as mean ± SD, n=2.

    Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry, Cell Culture, Real-time Polymerase Chain Reaction

    47) Product Images from "Notch signaling facilitates hepatitis B virus covalently closed circular DNA transcription via cAMP response element-binding protein with E3 ubiquitin ligase-modulation"

    Article Title: Notch signaling facilitates hepatitis B virus covalently closed circular DNA transcription via cAMP response element-binding protein with E3 ubiquitin ligase-modulation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-38139-5

    E3 ubiquitin ligases regulate Notch intracellular domain and intranuclear HBV DNA levels. ( A ) Levels of E3 ubiquitin ligase genes (ITCH and NUMB) were upregulated and NOTCH1 was downregulated in HepG2.2.15.7 and HepAD38 cells transfected with Jagged1 shRNA. ( B ) E3 ubiquitin ligase gene expression was upregulated and Notch1 expression was downregulated in HepG2.2.15.7 and HepAD38 cells after 20 µM DAPT treatment for 120 h. ( C ) Western blotting confirmed the protein level trends of the E3 ubiquitin ligases and Notch-related markers in Jagged1 shRNA-transfected cells. Relative pixel intensities (right) were normalized to the GAPDH pixel intensity (Control-shRNA = 1). ( D ) Western blotting revealed the same protein level trends of the E3 ubiquitin ligases and Notch-related markers after 20 µM DAPT treatment. Relative pixel intensities (right) were normalized to the GAPDH pixel intensity (DMSO = 1). ( E ) Gene expression levels of ITCH and NUMB were decreased in HBV-replicating cell lines. ( F ) Gene expression levels of ITCH and NUMB were significantly inhibited by ITCH and NUMB-siRNA. Blocked expression of ITCH and NUMB by siRNA significantly increased the HBV cccDNA level, as determined by RTD-PCR ( G ) and Southern blotting ( H ). ( I ) NICD protein expression was increased after siITCH and siNUMB transfection. Relative pixel intensities (right) were normalized to the GAPDH pixel intensity (Control-siRNA = 1). Quantitative gene expression data represent the mean ± standard deviation of three independent experiments and were normalized to the expression levels of human GAPDH. * P
    Figure Legend Snippet: E3 ubiquitin ligases regulate Notch intracellular domain and intranuclear HBV DNA levels. ( A ) Levels of E3 ubiquitin ligase genes (ITCH and NUMB) were upregulated and NOTCH1 was downregulated in HepG2.2.15.7 and HepAD38 cells transfected with Jagged1 shRNA. ( B ) E3 ubiquitin ligase gene expression was upregulated and Notch1 expression was downregulated in HepG2.2.15.7 and HepAD38 cells after 20 µM DAPT treatment for 120 h. ( C ) Western blotting confirmed the protein level trends of the E3 ubiquitin ligases and Notch-related markers in Jagged1 shRNA-transfected cells. Relative pixel intensities (right) were normalized to the GAPDH pixel intensity (Control-shRNA = 1). ( D ) Western blotting revealed the same protein level trends of the E3 ubiquitin ligases and Notch-related markers after 20 µM DAPT treatment. Relative pixel intensities (right) were normalized to the GAPDH pixel intensity (DMSO = 1). ( E ) Gene expression levels of ITCH and NUMB were decreased in HBV-replicating cell lines. ( F ) Gene expression levels of ITCH and NUMB were significantly inhibited by ITCH and NUMB-siRNA. Blocked expression of ITCH and NUMB by siRNA significantly increased the HBV cccDNA level, as determined by RTD-PCR ( G ) and Southern blotting ( H ). ( I ) NICD protein expression was increased after siITCH and siNUMB transfection. Relative pixel intensities (right) were normalized to the GAPDH pixel intensity (Control-siRNA = 1). Quantitative gene expression data represent the mean ± standard deviation of three independent experiments and were normalized to the expression levels of human GAPDH. * P

    Techniques Used: Transfection, shRNA, Expressing, Western Blot, Polymerase Chain Reaction, Southern Blot, Standard Deviation

    Proteasome inhibitor (MG-132) promotes Jagged1/Notch1-dependent signal transduction and intrahepatic HBV cccDNA accumulation. ( A ) IC50 of MG-132 after 48 h treatment of HepG2 cells. ( B ) RTD-PCR analysis of the cccDNA level in HepG2.2.15.7 and HepAD38 cells transfected with Jagged1 shRNA with or without MG-132 (0.5 µM) treatment for 48 h. Results are expressed as the number of cccDNA copies per genomic μg (mean ± standard deviation) from three independent experiments. * P
    Figure Legend Snippet: Proteasome inhibitor (MG-132) promotes Jagged1/Notch1-dependent signal transduction and intrahepatic HBV cccDNA accumulation. ( A ) IC50 of MG-132 after 48 h treatment of HepG2 cells. ( B ) RTD-PCR analysis of the cccDNA level in HepG2.2.15.7 and HepAD38 cells transfected with Jagged1 shRNA with or without MG-132 (0.5 µM) treatment for 48 h. Results are expressed as the number of cccDNA copies per genomic μg (mean ± standard deviation) from three independent experiments. * P

    Techniques Used: Transduction, Polymerase Chain Reaction, Transfection, shRNA, Standard Deviation

    Notch inhibition markedly suppresses hepatis B virus (HBV) covalently closed circular DNA (cccDNA). ( A ) HBV Hirt DNA was extracted from HepG2.2.15.7 (i) and HepAD38 (ii) cells and detected by Southern blotting under the condition of no treatment, 85 °C for 5 min, or 85 °C for 5 min plus EcoRI 2 h digestion. (iii) EcoRI digestion of HepG2.2.15.7 cells for 2 h and 16 h after 85 °C treatment. ( B ) Jagged1 mRNA and protein levels were significantly decreased after transfection with Jagged1 shRNA, as detected by RTD-PCR (upper) and western blotting (below). ( C ) RTD-PCR quantification of cccDNA accumulation in HepG2.2.15.7 and HepAD38 cells transfected with lentiviruses encoding Jagged1 short hairpin RNA (shRNA). ( D ) Southern blot analysis confirming decreased cccDNA levels in HepG2.2.15.7 and HepAD38 cells transfected with Jagged1 shRNA. ( E ) No significant change in cell numbers at 0 and 120 h between 20 µM DMSO and DAPT treatment. ( F ) RTD-PCR quantification of cccDNA accumulation in HepG2.2.15.7 and HepAD38 cells administered 20 µM DAPT. ( G ) Southern blot analysis confirming decreased cccDNA levels in HepG2.2.15.7 and HepAD38 cells treated with 20 µM DAPT. cccDNA results are expressed as the number of cccDNA copies per genomic μg (mean ± standard deviation) from three independent experiments. * P
    Figure Legend Snippet: Notch inhibition markedly suppresses hepatis B virus (HBV) covalently closed circular DNA (cccDNA). ( A ) HBV Hirt DNA was extracted from HepG2.2.15.7 (i) and HepAD38 (ii) cells and detected by Southern blotting under the condition of no treatment, 85 °C for 5 min, or 85 °C for 5 min plus EcoRI 2 h digestion. (iii) EcoRI digestion of HepG2.2.15.7 cells for 2 h and 16 h after 85 °C treatment. ( B ) Jagged1 mRNA and protein levels were significantly decreased after transfection with Jagged1 shRNA, as detected by RTD-PCR (upper) and western blotting (below). ( C ) RTD-PCR quantification of cccDNA accumulation in HepG2.2.15.7 and HepAD38 cells transfected with lentiviruses encoding Jagged1 short hairpin RNA (shRNA). ( D ) Southern blot analysis confirming decreased cccDNA levels in HepG2.2.15.7 and HepAD38 cells transfected with Jagged1 shRNA. ( E ) No significant change in cell numbers at 0 and 120 h between 20 µM DMSO and DAPT treatment. ( F ) RTD-PCR quantification of cccDNA accumulation in HepG2.2.15.7 and HepAD38 cells administered 20 µM DAPT. ( G ) Southern blot analysis confirming decreased cccDNA levels in HepG2.2.15.7 and HepAD38 cells treated with 20 µM DAPT. cccDNA results are expressed as the number of cccDNA copies per genomic μg (mean ± standard deviation) from three independent experiments. * P

    Techniques Used: Inhibition, Southern Blot, Transfection, shRNA, Polymerase Chain Reaction, Western Blot, Standard Deviation

    48) Product Images from "Transcriptome-wide analysis uncovers the targets of the RNA-binding protein MSI2 and effects of MSI2's RNA-binding activity on IL-6 signaling"

    Article Title: Transcriptome-wide analysis uncovers the targets of the RNA-binding protein MSI2 and effects of MSI2's RNA-binding activity on IL-6 signaling

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.002243

    MSI2 regulates IL6ST transcript levels. A , expression correlation measured by RNA-Seq between IL6ST and MSI2 in Mock and MSI2 KD samples. B , quantitative real-time PCR of differential gene regulation of IL6ST upon MSI2 siRNA–treated cells and untreated cells. qPCR data were analyzed using two-way ANOVA and presented as mean ± S.D. (***, p
    Figure Legend Snippet: MSI2 regulates IL6ST transcript levels. A , expression correlation measured by RNA-Seq between IL6ST and MSI2 in Mock and MSI2 KD samples. B , quantitative real-time PCR of differential gene regulation of IL6ST upon MSI2 siRNA–treated cells and untreated cells. qPCR data were analyzed using two-way ANOVA and presented as mean ± S.D. (***, p

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

    49) Product Images from "P66Shc, a key regulator of metabolism and mitochondrial ROS production, is dysregulated by mouse embryo culture"

    Article Title: P66Shc, a key regulator of metabolism and mitochondrial ROS production, is dysregulated by mouse embryo culture

    Journal: Molecular Human Reproduction

    doi: 10.1093/molehr/gaw043

    p66Shc expression increases during mouse preimplantation development in vivo . ( A ) Quantitative real time RT-PCR (qRT-PCR) for p66Shc relative transcript abundance was performed on three replicates of pools of 20 embryos per stage. P66Shc relative transcript abundance significantly increases from eight cell to blastocyst-stage embryos ( n = 3, mean ± SEM, P = 0.0476 1W-ANOVA). ( B ) Immunoblotting for total p66Shc protein abundance was performed on three replicates of pools of 30–50 embryos per stage. P66Shc relative protein abundance increases from eight cell to blastocyst-stage embryos ( n = 3, mean ± SEM, P ).
    Figure Legend Snippet: p66Shc expression increases during mouse preimplantation development in vivo . ( A ) Quantitative real time RT-PCR (qRT-PCR) for p66Shc relative transcript abundance was performed on three replicates of pools of 20 embryos per stage. P66Shc relative transcript abundance significantly increases from eight cell to blastocyst-stage embryos ( n = 3, mean ± SEM, P = 0.0476 1W-ANOVA). ( B ) Immunoblotting for total p66Shc protein abundance was performed on three replicates of pools of 30–50 embryos per stage. P66Shc relative protein abundance increases from eight cell to blastocyst-stage embryos ( n = 3, mean ± SEM, P ).

    Techniques Used: Expressing, In Vivo, Quantitative RT-PCR

    50) Product Images from "Evolution of an insect immune barrier through horizontal gene transfer mediated by a parasitic wasp"

    Article Title: Evolution of an insect immune barrier through horizontal gene transfer mediated by a parasitic wasp

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007998

    Phagocytosis by Spodoptera littoralis larvae as affected by the presence in the plasma of the opsonizing factor Sl gasmin. RNAi mediated silencing of the gene Sl gasmin significantly reduced its transcription level, determined by qRT-PCR (A) and the presence of the encoded protein in the haemolymph plasma, determined by LC-MRM/MS (C), both under basal and immune challenged conditions. The presence of Sl gasmin in the incubation medium was essential to promote phagocytosis in vitro of fluorescent bacteria ( Escherichia coli ), as supported by the functional rescue of haemocytes unable to perform phagocytosis, obtained from Sl gasmin silenced larvae, when transferred to plasma of control larvae (B). The opsonizing role of Sl gasmin was demonstrated by LC-MRM/MS measurements of its amount recovered by proteolytic shaving of E . coli cells incubated in presence of haemolymph plasma (D). S . littoralis β-actin was used as reporter gene. The values reported are the mean ± SE. Different letters above each bar, in A and C, indicate significant differences ( P
    Figure Legend Snippet: Phagocytosis by Spodoptera littoralis larvae as affected by the presence in the plasma of the opsonizing factor Sl gasmin. RNAi mediated silencing of the gene Sl gasmin significantly reduced its transcription level, determined by qRT-PCR (A) and the presence of the encoded protein in the haemolymph plasma, determined by LC-MRM/MS (C), both under basal and immune challenged conditions. The presence of Sl gasmin in the incubation medium was essential to promote phagocytosis in vitro of fluorescent bacteria ( Escherichia coli ), as supported by the functional rescue of haemocytes unable to perform phagocytosis, obtained from Sl gasmin silenced larvae, when transferred to plasma of control larvae (B). The opsonizing role of Sl gasmin was demonstrated by LC-MRM/MS measurements of its amount recovered by proteolytic shaving of E . coli cells incubated in presence of haemolymph plasma (D). S . littoralis β-actin was used as reporter gene. The values reported are the mean ± SE. Different letters above each bar, in A and C, indicate significant differences ( P

    Techniques Used: Quantitative RT-PCR, Mass Spectrometry, Incubation, In Vitro, Functional Assay

    Relative expression level of Sl gasmin after microbial challenge. The transcription level of Sl gasmin , determined by qRT-PCR, was significantly up-regulated in Spodoptera littoralis larvae by injection of the Gram-positive bacterium Staphylococcus aureus , the Gram-negative bacterium Escherichia coli , and the yeast Saccharomyces cerevisiae . S . littoralis β-actin was used as reporter gene. Data points are the mean ± S.E. of 3 biological replicates. Different letters above each bar indicate significant differences ( P
    Figure Legend Snippet: Relative expression level of Sl gasmin after microbial challenge. The transcription level of Sl gasmin , determined by qRT-PCR, was significantly up-regulated in Spodoptera littoralis larvae by injection of the Gram-positive bacterium Staphylococcus aureus , the Gram-negative bacterium Escherichia coli , and the yeast Saccharomyces cerevisiae . S . littoralis β-actin was used as reporter gene. Data points are the mean ± S.E. of 3 biological replicates. Different letters above each bar indicate significant differences ( P

    Techniques Used: Expressing, Quantitative RT-PCR, Injection

    Relative quantification of bacterial load by qRT-PCR. Relative change over time of the bacterial load in Spodoptera littoralis larvae, exposed to control dsRNA (A) or Sl gasmin dsRNA (B) and fed with artificial diet on which a solution of Bacillus thuringiensis Cry1Ca toxin (2.7 μg/cm 2 ) was layered. The bacterial load in the haemolymph (H, red lines with empty circles) resulted significantly influenced by toxin treatment, both in control and silenced larvae, with these latter showing a much higher bacterial load increase over time (see S1 Table for statistics), whereas no significant changes were observed in the midgut (M).
    Figure Legend Snippet: Relative quantification of bacterial load by qRT-PCR. Relative change over time of the bacterial load in Spodoptera littoralis larvae, exposed to control dsRNA (A) or Sl gasmin dsRNA (B) and fed with artificial diet on which a solution of Bacillus thuringiensis Cry1Ca toxin (2.7 μg/cm 2 ) was layered. The bacterial load in the haemolymph (H, red lines with empty circles) resulted significantly influenced by toxin treatment, both in control and silenced larvae, with these latter showing a much higher bacterial load increase over time (see S1 Table for statistics), whereas no significant changes were observed in the midgut (M).

    Techniques Used: Quantitative RT-PCR

    Sl gasmin silencing in Spodoptera littoralis larvae. After oral administration of control or Sl g amin dsRNA during 4 th instar, the expression of Sl gamin , determined by qRT-PCR was significantly down-regulated until pupation. S . littoralis β-actin was used as reporter gene. The values reported are the mean ± S.E. (* P
    Figure Legend Snippet: Sl gasmin silencing in Spodoptera littoralis larvae. After oral administration of control or Sl g amin dsRNA during 4 th instar, the expression of Sl gamin , determined by qRT-PCR was significantly down-regulated until pupation. S . littoralis β-actin was used as reporter gene. The values reported are the mean ± S.E. (* P

    Techniques Used: Expressing, Quantitative RT-PCR

    Sl gasmin transcript level in different tissues of Spodoptera littoralis larvae. Sl gasmin relative expression, determined by qRT-PCR, was significantly higher in the haemocytes compared with the other tissues analyzed. S . littoralis β-actin was used as reporter gene. The values reported are the mean ± S.E. (* P
    Figure Legend Snippet: Sl gasmin transcript level in different tissues of Spodoptera littoralis larvae. Sl gasmin relative expression, determined by qRT-PCR, was significantly higher in the haemocytes compared with the other tissues analyzed. S . littoralis β-actin was used as reporter gene. The values reported are the mean ± S.E. (* P

    Techniques Used: Expressing, Quantitative RT-PCR

    51) Product Images from "Genetic manipulation of Staphylococcus aureus"

    Article Title: Genetic manipulation of Staphylococcus aureus

    Journal: Current protocols in microbiology

    doi: 10.1002/9780471729259.mc09c03s32

    Map of transposon bursa aurealis and plasmids used for delivery along with summary of steps involved in the mapping of insertion sites by inverse PCR. (A) Bursa aurealis (3.2 kbp), a mini-mariner transposable element, was cloned into pTS2, with a temperature-sensitive plasmid replicon (rep ts ) and chloramphenicol resistance gene cat to generate pBursa (7,383 bp). Bursa aurealis encompasses mariner terminal inverted repeats (TIR), R6K replication origin (oriV) for replication in E. coli , and erythromycin-resistance determinant ermC , an rRNA methylase that allows selection in both E. coli and S. aureus . The position of the most terminal site for the restriction enzyme Aci I is indicated as well as the site of hybridization and nucleotide sequence of primer Martn-F (F). Plasmid pFA545 (10,079 bp) encodes the mariner transposase tnp and is a derivative of pSPT181, a shuttle vector consisting of pSP64 with ampicillin resistance ( bla ) for replication and selection in E. coli , and pRN8103, a temperature-sensitive derivative of pT181 (rep ts ) and tetracycline-resistance marker ( tetB tetD ). The presence of rep ts and tetBD allows for replication of pFA545 in S. aureus and other Gram-positive bacteria. (B) Mapping insertion sites by inverse PCR. Genome DNA from a candidate mutant strain is isolated and digested with Aci I. Next, fragment self-ligation and inverse PCR are performed using DNA ligase and primers Martn-F (F) and Martn-ermR (R). PCR products are subjected to DNA sequence analysis using primer Martn-F (F).
    Figure Legend Snippet: Map of transposon bursa aurealis and plasmids used for delivery along with summary of steps involved in the mapping of insertion sites by inverse PCR. (A) Bursa aurealis (3.2 kbp), a mini-mariner transposable element, was cloned into pTS2, with a temperature-sensitive plasmid replicon (rep ts ) and chloramphenicol resistance gene cat to generate pBursa (7,383 bp). Bursa aurealis encompasses mariner terminal inverted repeats (TIR), R6K replication origin (oriV) for replication in E. coli , and erythromycin-resistance determinant ermC , an rRNA methylase that allows selection in both E. coli and S. aureus . The position of the most terminal site for the restriction enzyme Aci I is indicated as well as the site of hybridization and nucleotide sequence of primer Martn-F (F). Plasmid pFA545 (10,079 bp) encodes the mariner transposase tnp and is a derivative of pSPT181, a shuttle vector consisting of pSP64 with ampicillin resistance ( bla ) for replication and selection in E. coli , and pRN8103, a temperature-sensitive derivative of pT181 (rep ts ) and tetracycline-resistance marker ( tetB tetD ). The presence of rep ts and tetBD allows for replication of pFA545 in S. aureus and other Gram-positive bacteria. (B) Mapping insertion sites by inverse PCR. Genome DNA from a candidate mutant strain is isolated and digested with Aci I. Next, fragment self-ligation and inverse PCR are performed using DNA ligase and primers Martn-F (F) and Martn-ermR (R). PCR products are subjected to DNA sequence analysis using primer Martn-F (F).

    Techniques Used: Inverse PCR, Clone Assay, Plasmid Preparation, Selection, Hybridization, Sequencing, Marker, Mutagenesis, Isolation, Ligation, Polymerase Chain Reaction

    52) Product Images from "miR-424 coordinates multilayered regulation of cell cycle progression to promote esophageal squamous cell carcinoma cell proliferation"

    Article Title: miR-424 coordinates multilayered regulation of cell cycle progression to promote esophageal squamous cell carcinoma cell proliferation

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2018.10.043

    miR-424 promotes ESCC cell proliferation in vitro and in vivo . (a) qRT-PCR analyses showed the relative miR-424 expression levels in KYSE-410 and KYSE-510 ESCC cells and NE1 immortalized esophageal epithelial cells expressing the miR-424 precursor (lenti- miR-424 ) or control vectors. Data are presented as the mean ± SD of three independent experiments. **, p
    Figure Legend Snippet: miR-424 promotes ESCC cell proliferation in vitro and in vivo . (a) qRT-PCR analyses showed the relative miR-424 expression levels in KYSE-410 and KYSE-510 ESCC cells and NE1 immortalized esophageal epithelial cells expressing the miR-424 precursor (lenti- miR-424 ) or control vectors. Data are presented as the mean ± SD of three independent experiments. **, p

    Techniques Used: In Vitro, In Vivo, Quantitative RT-PCR, Expressing

    Characterization of the miR-424 promoter and transcriptional regulation of miR-424 by E2F1 during G1/S transition. (a) KYSE-410 and KYSE-510 cells were synchronized in G0/G1-phase by serum starvation and released. qRT-PCR analysis shows the expression kinetics of miR-424 , pri- miR-424 , and pre- miR-424 in KYSE-410 and KYSE-510 cells after release from G0/G1-phase for the indicated time points. Data are presented as the mean ± SD of three independent experiments. (b) A schematic illustration shows two binding sites for E2F1 in the putative promoter region of the miR-424 gene. Specific primers surrounding the promoter were designed. A fragment of the promoter region encompassing the wild-type or mutant E2F1 binding sites was cloned into a reporter vector. (c) Expression of E2F1 was silenced using siRNA (left panel). qRT-PCR showed changes in the expression kinetics of miR-424 , pri- miR-424 , and pre- miR-424 after knocking down E2F1 in KYSE-410 cells during G1/S transition (right panel). Data are presented as the mean ± SD of three independent experiments. *, p
    Figure Legend Snippet: Characterization of the miR-424 promoter and transcriptional regulation of miR-424 by E2F1 during G1/S transition. (a) KYSE-410 and KYSE-510 cells were synchronized in G0/G1-phase by serum starvation and released. qRT-PCR analysis shows the expression kinetics of miR-424 , pri- miR-424 , and pre- miR-424 in KYSE-410 and KYSE-510 cells after release from G0/G1-phase for the indicated time points. Data are presented as the mean ± SD of three independent experiments. (b) A schematic illustration shows two binding sites for E2F1 in the putative promoter region of the miR-424 gene. Specific primers surrounding the promoter were designed. A fragment of the promoter region encompassing the wild-type or mutant E2F1 binding sites was cloned into a reporter vector. (c) Expression of E2F1 was silenced using siRNA (left panel). qRT-PCR showed changes in the expression kinetics of miR-424 , pri- miR-424 , and pre- miR-424 after knocking down E2F1 in KYSE-410 cells during G1/S transition (right panel). Data are presented as the mean ± SD of three independent experiments. *, p

    Techniques Used: Quantitative RT-PCR, Expressing, Binding Assay, Mutagenesis, Clone Assay, Plasmid Preparation

    53) Product Images from "Fe(III) Is Essential for Porcine Embryonic Development via Mitochondrial Function Maintenance"

    Article Title: Fe(III) Is Essential for Porcine Embryonic Development via Mitochondrial Function Maintenance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0130791

    Expression of apoptosis related miRNAs in porcine blastocysts cultured for seven days. Apoptosis-related miRNAs were analyzed by TaqMan real-time RT-PCR. Black bar, control group; white bar, DFM 0.5 μM; gray bar, DFM 5.0 μM. * P
    Figure Legend Snippet: Expression of apoptosis related miRNAs in porcine blastocysts cultured for seven days. Apoptosis-related miRNAs were analyzed by TaqMan real-time RT-PCR. Black bar, control group; white bar, DFM 0.5 μM; gray bar, DFM 5.0 μM. * P

    Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR

    54) Product Images from "A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii"

    Article Title: A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii

    Journal: Annals of Laboratory Medicine

    doi: 10.3343/alm.2020.40.1.27

    PCR-RFLP patterns obtained following digestion with Hinf I or Xmn I for gyrA and parC . Lanes 1 to 3 and 4 to 6 show PCR-RFLP results for gyrA and parC , respectively. Lane: M, 20 bp DNA ladder marker. (A) PCR-RFLP results for A. baumannii ATCC19606. Lanes: 1, undigested (143 bp); 2, Hinf I-digestion (103 bp and 40 bp); 3, Xmn I-digestion (122 bp and 21 bp); 4, undigested (120 bp); 5, Hinf I-digestion (85 bp and 35 bp); and 6, Xmn I-digestion (104 bp and 16 bp). (B) PCR-RFLP results for the representative FQ-resistant A. baumannii strain possessing mutations in gyrA (83) and parC (80). Lanes: 1, undigested (143 bp); 2, Hinf I-digestion (143 bp); 3, Xmn I-digestion (122 bp and 21 bp); 4, undigested (120 bp); 5, Hinf I-digestion (120 bp); and 6, Xmn I-digestion (104 bp and 16 bp). Abbreviations: FQ, fluoroquinolone; RFLP, restriction fragment length polymorphism.
    Figure Legend Snippet: PCR-RFLP patterns obtained following digestion with Hinf I or Xmn I for gyrA and parC . Lanes 1 to 3 and 4 to 6 show PCR-RFLP results for gyrA and parC , respectively. Lane: M, 20 bp DNA ladder marker. (A) PCR-RFLP results for A. baumannii ATCC19606. Lanes: 1, undigested (143 bp); 2, Hinf I-digestion (103 bp and 40 bp); 3, Xmn I-digestion (122 bp and 21 bp); 4, undigested (120 bp); 5, Hinf I-digestion (85 bp and 35 bp); and 6, Xmn I-digestion (104 bp and 16 bp). (B) PCR-RFLP results for the representative FQ-resistant A. baumannii strain possessing mutations in gyrA (83) and parC (80). Lanes: 1, undigested (143 bp); 2, Hinf I-digestion (143 bp); 3, Xmn I-digestion (122 bp and 21 bp); 4, undigested (120 bp); 5, Hinf I-digestion (120 bp); and 6, Xmn I-digestion (104 bp and 16 bp). Abbreviations: FQ, fluoroquinolone; RFLP, restriction fragment length polymorphism.

    Techniques Used: Polymerase Chain Reaction, Marker

    55) Product Images from "The osteopetrotic mutation toothless (tl) is a loss-of-function frameshift mutation in the rat Csf1 gene: Evidence of a crucial role for CSF-1 in osteoclastogenesis and endochondral ossification"

    Article Title: The osteopetrotic mutation toothless (tl) is a loss-of-function frameshift mutation in the rat Csf1 gene: Evidence of a crucial role for CSF-1 in osteoclastogenesis and endochondral ossification

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

    doi: 10.1073/pnas.202332999

    Alternative splicing of rat CSF-1 mRNA reveals no skipping of tl mutation-containing region. CSF-1 cDNAs were amplified from rat cDNA, separated in an agarose gel, blotted, and probed with 32 P-labeled rat CSF-1 cDNA. Lanes 1–8 represent skeletal muscle, liver, kidney, lung, brain, testis, pancreas, and heart, respectively. ( A ) RT-PCR product encompassing the complete coding sequence of rat CSF-1. Two alternative transcripts of 0.96 and 1.91 kb, respectively, can be distinguished. The shorter fragment can be explained by alternative splicing deletion of the major part of exon 6, as has been described in mouse and human. ( B ) PCR product containing the first 3 exons of rat CSF-1 cDNA. Only one transcript can be distinguished, indicating no alternative splicing in the 5′ part of the transcript that could bypass the tl mutation.
    Figure Legend Snippet: Alternative splicing of rat CSF-1 mRNA reveals no skipping of tl mutation-containing region. CSF-1 cDNAs were amplified from rat cDNA, separated in an agarose gel, blotted, and probed with 32 P-labeled rat CSF-1 cDNA. Lanes 1–8 represent skeletal muscle, liver, kidney, lung, brain, testis, pancreas, and heart, respectively. ( A ) RT-PCR product encompassing the complete coding sequence of rat CSF-1. Two alternative transcripts of 0.96 and 1.91 kb, respectively, can be distinguished. The shorter fragment can be explained by alternative splicing deletion of the major part of exon 6, as has been described in mouse and human. ( B ) PCR product containing the first 3 exons of rat CSF-1 cDNA. Only one transcript can be distinguished, indicating no alternative splicing in the 5′ part of the transcript that could bypass the tl mutation.

    Techniques Used: Mutagenesis, Amplification, Agarose Gel Electrophoresis, Labeling, Reverse Transcription Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction

    56) Product Images from "Changes in the Expression of Human Cell Division Autoantigen-1 Influence Toxoplasmagondii Growth and Development"

    Article Title: Changes in the Expression of Human Cell Division Autoantigen-1 Influence Toxoplasmagondii Growth and Development

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0020105

    siRNAs against CDA1 Antagonize Compound 1 Inhibition of Parasite Growth (A) HFF cells transfected with the siRNA SmartPool against CDA1 and co-treated with Compound 1 prior to parasite infection allow normal parasite growth (black boxes) and BAG1 expression (dark bars). Compare growth in untreated controls (black circles) with growth (grey boxes) and BAG1 expression (light bars) in Compound 1–treated cells. (B) Concurrent with the restoration of normal parasite growth and BAG1 expression, we observed the loss of Compound 1–induced CDA1 mRNAs as measured by RT-PCR of total RNA at 24, 48 following transfection with siRNA and Compound 1 pretreatment. Lamin A/C controls demonstrate siRNA-mediated knockdown of individual genes. Transfection with siRNAs against Lamin A/C, with nonspecific siRNAs or with LipofectAMINE alone, was unable to antagonize Compound 1 inhibition of parasite growth or induction of BAG1 expression (results not shown). RT-PCR primers specific to GAPDH were used as a control for RNA quality. RT-PCR experiment: C = Compound 1 pretreatment (3 h/3 μM) only; E = HFF cells pretreated with both Compound 1 and siRNA(s) against either CDA1 or Lamin A/C. Protein blot: L = HFF cells pretreated with LipofectAMINE, but without siRNAs; si = HFF cells pretreated with siRNAs against either CDA1 or Lamin A/C, but without transfection reagent (LipofectAMINE); E = same as above. (C) Co-transfection of HFF cells with siRNAs 1 to 4 separately and then treatment with Compound 1 prior to parasite infection demonstrate that siRNAs 2 and 3 were responsible for antagonizing the Compound 1–induced inhibition of parasite growth as measured by average parasites per vacuole at 48 h postinfection. Compare light bars for siRNAs 2 and 3 with untreated parasites (labeled no siRNA), and also with Compound 1–treated parasites (labeled Compound 1). siRNAs 1 and 4 do not significantly antagonize the Compound 1–induced inhibition of parasite replication.
    Figure Legend Snippet: siRNAs against CDA1 Antagonize Compound 1 Inhibition of Parasite Growth (A) HFF cells transfected with the siRNA SmartPool against CDA1 and co-treated with Compound 1 prior to parasite infection allow normal parasite growth (black boxes) and BAG1 expression (dark bars). Compare growth in untreated controls (black circles) with growth (grey boxes) and BAG1 expression (light bars) in Compound 1–treated cells. (B) Concurrent with the restoration of normal parasite growth and BAG1 expression, we observed the loss of Compound 1–induced CDA1 mRNAs as measured by RT-PCR of total RNA at 24, 48 following transfection with siRNA and Compound 1 pretreatment. Lamin A/C controls demonstrate siRNA-mediated knockdown of individual genes. Transfection with siRNAs against Lamin A/C, with nonspecific siRNAs or with LipofectAMINE alone, was unable to antagonize Compound 1 inhibition of parasite growth or induction of BAG1 expression (results not shown). RT-PCR primers specific to GAPDH were used as a control for RNA quality. RT-PCR experiment: C = Compound 1 pretreatment (3 h/3 μM) only; E = HFF cells pretreated with both Compound 1 and siRNA(s) against either CDA1 or Lamin A/C. Protein blot: L = HFF cells pretreated with LipofectAMINE, but without siRNAs; si = HFF cells pretreated with siRNAs against either CDA1 or Lamin A/C, but without transfection reagent (LipofectAMINE); E = same as above. (C) Co-transfection of HFF cells with siRNAs 1 to 4 separately and then treatment with Compound 1 prior to parasite infection demonstrate that siRNAs 2 and 3 were responsible for antagonizing the Compound 1–induced inhibition of parasite growth as measured by average parasites per vacuole at 48 h postinfection. Compare light bars for siRNAs 2 and 3 with untreated parasites (labeled no siRNA), and also with Compound 1–treated parasites (labeled Compound 1). siRNAs 1 and 4 do not significantly antagonize the Compound 1–induced inhibition of parasite replication.

    Techniques Used: Inhibition, Transfection, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Cotransfection, Labeling

    57) Product Images from "Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2"

    Article Title: Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.00967

    Detection of DM2 mutation at DNA, expression of CNBP gene and RNA level during differentiation. (A) Percentages of nuclear foci (1–4 or > 5) in hiPSCs and NP showing the increase of foci number along their differentiation process. (B) LR-PCR followed by hybridization with a (CTG) 5 -radioactively labeled probe on DNA extracted from DM2 hiPSCs and NPs, arrows indicated CNBP normal and expanded alleles. (C) RT-qPCR assay for CNBP expression in DM2 hiPSCs and NPs. β-actin is used as reference gene.
    Figure Legend Snippet: Detection of DM2 mutation at DNA, expression of CNBP gene and RNA level during differentiation. (A) Percentages of nuclear foci (1–4 or > 5) in hiPSCs and NP showing the increase of foci number along their differentiation process. (B) LR-PCR followed by hybridization with a (CTG) 5 -radioactively labeled probe on DNA extracted from DM2 hiPSCs and NPs, arrows indicated CNBP normal and expanded alleles. (C) RT-qPCR assay for CNBP expression in DM2 hiPSCs and NPs. β-actin is used as reference gene.

    Techniques Used: Mutagenesis, Expressing, Polymerase Chain Reaction, Hybridization, CTG Assay, Labeling, Quantitative RT-PCR

    58) Product Images from "Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2"

    Article Title: Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.00967

    Detection of DM2 mutation at DNA, expression of CNBP gene and RNA level during differentiation. (A) Percentages of nuclear foci (1–4 or > 5) in hiPSCs and NP showing the increase of foci number along their differentiation process. (B) LR-PCR followed by hybridization with a (CTG) 5 -radioactively labeled probe on DNA extracted from DM2 hiPSCs and NPs, arrows indicated CNBP normal and expanded alleles. (C) RT-qPCR assay for CNBP expression in DM2 hiPSCs and NPs. β-actin is used as reference gene.
    Figure Legend Snippet: Detection of DM2 mutation at DNA, expression of CNBP gene and RNA level during differentiation. (A) Percentages of nuclear foci (1–4 or > 5) in hiPSCs and NP showing the increase of foci number along their differentiation process. (B) LR-PCR followed by hybridization with a (CTG) 5 -radioactively labeled probe on DNA extracted from DM2 hiPSCs and NPs, arrows indicated CNBP normal and expanded alleles. (C) RT-qPCR assay for CNBP expression in DM2 hiPSCs and NPs. β-actin is used as reference gene.

    Techniques Used: Mutagenesis, Expressing, Polymerase Chain Reaction, Hybridization, CTG Assay, Labeling, Quantitative RT-PCR

    59) Product Images from "The absence of dystrophin brain isoform expression in healthy human heart ventricles explains the pathogenesis of 5' X-linked dilated cardiomyopathy"

    Article Title: The absence of dystrophin brain isoform expression in healthy human heart ventricles explains the pathogenesis of 5' X-linked dilated cardiomyopathy

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-13-20

    a. Hybridization of dystrophin B isoform RT-PCR (oligonucleotides within B exons 1 and 6; expected product: 514 bp) with an internal oligo probe (within exon 2) that confirmed its expression in both Atria (lanes 2 and 4) and in the control brain (lane 6), and its absence in the Ventricles and Total Heart . b Levels of dystrophin M transcript expression (2-ΔΔCT values) using the skeletal muscle as internal calibrator and actin as reference gene in: Skeletal Muscle, Total heart, Right Atrium, Right Ventricle, Left Atrium, Left Ventricle, Sinoatrial (SA) Node, Atrioventricular (AV) Node, Bundle of His and Purkinje Fibres. The M isoform was expressed in all the samples studied, featuring the lowest score in Brain. c Levels of dystrophin B transcript expression (2-ΔΔCT values) using the brain as internal calibrator and actin as reference gene in the same samples. In both the ventricles the B amplification threshold cycle (CT) was undetermined, as well as in the total heart and in the conduction system structures. In the atria the score obtained was higher than that in skeletal muscle, in particular in the left atrium.
    Figure Legend Snippet: a. Hybridization of dystrophin B isoform RT-PCR (oligonucleotides within B exons 1 and 6; expected product: 514 bp) with an internal oligo probe (within exon 2) that confirmed its expression in both Atria (lanes 2 and 4) and in the control brain (lane 6), and its absence in the Ventricles and Total Heart . b Levels of dystrophin M transcript expression (2-ΔΔCT values) using the skeletal muscle as internal calibrator and actin as reference gene in: Skeletal Muscle, Total heart, Right Atrium, Right Ventricle, Left Atrium, Left Ventricle, Sinoatrial (SA) Node, Atrioventricular (AV) Node, Bundle of His and Purkinje Fibres. The M isoform was expressed in all the samples studied, featuring the lowest score in Brain. c Levels of dystrophin B transcript expression (2-ΔΔCT values) using the brain as internal calibrator and actin as reference gene in the same samples. In both the ventricles the B amplification threshold cycle (CT) was undetermined, as well as in the total heart and in the conduction system structures. In the atria the score obtained was higher than that in skeletal muscle, in particular in the left atrium.

    Techniques Used: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification

    60) Product Images from "Papillomavirus Genomes Associate with BRD4 to Replicate at Fragile Sites in the Host Genome"

    Article Title: Papillomavirus Genomes Associate with BRD4 to Replicate at Fragile Sites in the Host Genome

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004117

    Persistent E2 binding correlates with histone acetylation through CREBBP/EP300 HAT activity. A. Mitotic chromatin was isolated from C-33-1E2 cells and immunoprecipitated with control serum or specific antibodies to E2, BRD4, H3K56ac, H4K8ac, H3K4me1, H3K4me2, H3K4me3, and histone H3. ChIP DNA was analyzed by Q-PCR for specific PEB-BLOC regions (listed in Table S9 ). Average values and STDEV are shown for three independent experiments on four non-E2 binding regions, four active promoters, and six PEB-BLOCs. B. ChIP-chip analysis of E2, BRD4, H4K8ac, and H3K4me1 binding in C-33-1E2 cells. Chromatin was prepared from asynchronous cells and isolated using antibodies against E2, BRD4, acH4K8, and H3Kme14. ChIP DNA was hybridized to one HD microarray chip (Nimblegen). The binding profile for E2, BRD4, and histones on chromosome 3 and 4 is shown. Broad regions of enriched binding were defined computationally and are shown in red and are listed in Table S4 . C. Venn diagram showing the overlap among the enriched binding regions defined in B. D. C-33-1E2 cells were treated with CREBBP, EP300 or KAT5 siRNA for 3 days. Cells were stained by immunofluorescence for anti-BRD4 (green), anti-H4K8ac (red), cellular DNA (blue), and anti-CREBBP, -EP300, or -KAT5 antibodies (cyan). The bar chart to the right shows quantification of BRD4 speckle formation in these Interphase cells were analyzed for. Average values and STDEV were calculated for three independent experiments (75–150 cells counted per experiment).
    Figure Legend Snippet: Persistent E2 binding correlates with histone acetylation through CREBBP/EP300 HAT activity. A. Mitotic chromatin was isolated from C-33-1E2 cells and immunoprecipitated with control serum or specific antibodies to E2, BRD4, H3K56ac, H4K8ac, H3K4me1, H3K4me2, H3K4me3, and histone H3. ChIP DNA was analyzed by Q-PCR for specific PEB-BLOC regions (listed in Table S9 ). Average values and STDEV are shown for three independent experiments on four non-E2 binding regions, four active promoters, and six PEB-BLOCs. B. ChIP-chip analysis of E2, BRD4, H4K8ac, and H3K4me1 binding in C-33-1E2 cells. Chromatin was prepared from asynchronous cells and isolated using antibodies against E2, BRD4, acH4K8, and H3Kme14. ChIP DNA was hybridized to one HD microarray chip (Nimblegen). The binding profile for E2, BRD4, and histones on chromosome 3 and 4 is shown. Broad regions of enriched binding were defined computationally and are shown in red and are listed in Table S4 . C. Venn diagram showing the overlap among the enriched binding regions defined in B. D. C-33-1E2 cells were treated with CREBBP, EP300 or KAT5 siRNA for 3 days. Cells were stained by immunofluorescence for anti-BRD4 (green), anti-H4K8ac (red), cellular DNA (blue), and anti-CREBBP, -EP300, or -KAT5 antibodies (cyan). The bar chart to the right shows quantification of BRD4 speckle formation in these Interphase cells were analyzed for. Average values and STDEV were calculated for three independent experiments (75–150 cells counted per experiment).

    Techniques Used: Binding Assay, HAT Assay, Activity Assay, Isolation, Immunoprecipitation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Microarray, Staining, Immunofluorescence

    BRD4 is essential for persistent HPV1 E2 binding to host mitotic chromatin. A. E2 expression was induced in asynchronous C-33 cells expressing either wild-type or R37A/I73A E2 proteins. Chromatin was isolated with FLAG antibodies (against E2) or with BRD4 antiserum. ChIP DNA was quantitated by Q-PCR using primers specific for the PEB-BLOCs listed. Average values and STDEV were calculated from two independent experiments. B. The location of E2 wild-type or E2 R37A/I73A (green) and BRD4 (red) as detected by immunofluorescence. Cellular DNA is counterstained with DAPI in blue. Approximately 50 mitotic cells were analyzed for E2 and BRD4 chromosomal speckles. Average values and STDEV were calculated for three independent experiments. C. C-33-1E2 cells were treated with BRD4 siRNA for 3 days and stained for E2 (green), BRD4 (red) and cellular DNA (blue). Approximately 50 mitotic cells were analyzed for E2 and BRD4 chromosomal speckles. Average values and STDEV were calculated for three independent experiments. D. C-33 cells expressing HPV1 E2 were treated with DMSO, GSK525762 + (GSK+), or GSK525762 − (GSK−), for 24 h and E2 expression was induced for 4 h before fixation. Chromatin was isolated with FLAG M2 or with BRD4 immune serum. ChIP DNA was quantitated by Q-PCR using primers specific for the PEB-BLOCs shown. Average values and STDEV were calculated from two independent experiments. E. C-33-1E2 cells were treated with DMSO, GSK525762 + (GSK+), or GSK525762 − (GSK−), for 24 h and E2 expression was induced before fixation. Cells were stained for E2 (green), BRD4 (red) and cellular DNA (blue). Greater than 50 interphase cells were analyzed for E2 and BRD4 colocalization. Average values and STDEV were calculated for three independent experiments.
    Figure Legend Snippet: BRD4 is essential for persistent HPV1 E2 binding to host mitotic chromatin. A. E2 expression was induced in asynchronous C-33 cells expressing either wild-type or R37A/I73A E2 proteins. Chromatin was isolated with FLAG antibodies (against E2) or with BRD4 antiserum. ChIP DNA was quantitated by Q-PCR using primers specific for the PEB-BLOCs listed. Average values and STDEV were calculated from two independent experiments. B. The location of E2 wild-type or E2 R37A/I73A (green) and BRD4 (red) as detected by immunofluorescence. Cellular DNA is counterstained with DAPI in blue. Approximately 50 mitotic cells were analyzed for E2 and BRD4 chromosomal speckles. Average values and STDEV were calculated for three independent experiments. C. C-33-1E2 cells were treated with BRD4 siRNA for 3 days and stained for E2 (green), BRD4 (red) and cellular DNA (blue). Approximately 50 mitotic cells were analyzed for E2 and BRD4 chromosomal speckles. Average values and STDEV were calculated for three independent experiments. D. C-33 cells expressing HPV1 E2 were treated with DMSO, GSK525762 + (GSK+), or GSK525762 − (GSK−), for 24 h and E2 expression was induced for 4 h before fixation. Chromatin was isolated with FLAG M2 or with BRD4 immune serum. ChIP DNA was quantitated by Q-PCR using primers specific for the PEB-BLOCs shown. Average values and STDEV were calculated from two independent experiments. E. C-33-1E2 cells were treated with DMSO, GSK525762 + (GSK+), or GSK525762 − (GSK−), for 24 h and E2 expression was induced before fixation. Cells were stained for E2 (green), BRD4 (red) and cellular DNA (blue). Greater than 50 interphase cells were analyzed for E2 and BRD4 colocalization. Average values and STDEV were calculated for three independent experiments.

    Techniques Used: Binding Assay, Expressing, Isolation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunofluorescence, Staining

    61) Product Images from "Downstream Gene Activation of the Receptor ALX by the Agonist Annexin A1"

    Article Title: Downstream Gene Activation of the Receptor ALX by the Agonist Annexin A1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0012771

    Transfection of ALX receptor into HEK293 cells. A ) Generation of the HEK293 clones. pRc/CMV expression vector used to transfect ALX receptor into HEK293 cells to produce stable cell lines. Details of the full length protein sequence for the ALX receptor are also shown. B ) Representative expression of FPR receptors by PCR visualised on a 1% agarose gel: CMV, empty vector transfected cells; ALX, annexin A1 receptor transfected cells; (+) human neutrophil cDNA positive control; (−) water replaced cDNA negative control. In all experiments results are representative of three separate experiments with similar results. C ) Specific ALX receptor immunoreactive protein cell surface expression was determined using flow cytometry and compared to CMV stably transfected HEK293 cells.
    Figure Legend Snippet: Transfection of ALX receptor into HEK293 cells. A ) Generation of the HEK293 clones. pRc/CMV expression vector used to transfect ALX receptor into HEK293 cells to produce stable cell lines. Details of the full length protein sequence for the ALX receptor are also shown. B ) Representative expression of FPR receptors by PCR visualised on a 1% agarose gel: CMV, empty vector transfected cells; ALX, annexin A1 receptor transfected cells; (+) human neutrophil cDNA positive control; (−) water replaced cDNA negative control. In all experiments results are representative of three separate experiments with similar results. C ) Specific ALX receptor immunoreactive protein cell surface expression was determined using flow cytometry and compared to CMV stably transfected HEK293 cells.

    Techniques Used: Transfection, Clone Assay, Expressing, Plasmid Preparation, Stable Transfection, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Negative Control, Flow Cytometry, Cytometry

    62) Product Images from "Role of intragenic binding of cAMP responsive protein (CRP) in regulation of the succinate dehydrogenase genes Rv0249c-Rv0247c in TB complex mycobacteria"

    Article Title: Role of intragenic binding of cAMP responsive protein (CRP) in regulation of the succinate dehydrogenase genes Rv0249c-Rv0247c in TB complex mycobacteria

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv420

    CRP binds and regulates at the Rv0250c locus. ( A ) Organization of the region. ∼60nt of the 3′ end of hsp are shown on the far left. The top bar shows approximate distance in nt. Gray arrows represent the anti-sense ncRNAs IG200 (ncRv10250) and IG199 (ncRv10249), identified by Arnvig et al . ( 72 , 75 ). ( B ) ß-galactosidase assay of the individual promoter fusion constructs in M. bovis BCG wild type (black bar) and crp background (hashed bars). ( C ) Purified CRP Mt 's ability to bind to either site 1 or 2 in the upstream region of Rv0250c or sites 3 or 4 in the upstream region of Rv0249c. ( D ) Contribution of sites 2 and 3 to expression. Black bars, wild type, hashed bars, replaced promoter fusion construct at site 2 or site 3 as indicated by x-axis. ( E ) RT-PCR using cDNA generated in the absence (−) or presence (+) of reverse transcriptase. Heat killed genomic DNA from M. tuberculosis H37Rv (H). Corresponding products are indicated in panel (A). ( F ) Summary of data. Small arrows above sites 2 and 4 indicate the positions of the binding sites relative to the transcriptional start sites (tss) as published ( 71 ). Binding site 1 is centered 64 nt from the +1 of Rv0250c, while the center of binding site 3 is 67.5 nt from the +1 of Rv0249c. Sites 2 and 4 overlap tss's for Rv0250c and Rv0249c promoters, respectively. Small arrows under each locus indicate promoter fragments used in these studies. NT not tested. Shown are the means with standard deviations from three independent experiments. (*, P
    Figure Legend Snippet: CRP binds and regulates at the Rv0250c locus. ( A ) Organization of the region. ∼60nt of the 3′ end of hsp are shown on the far left. The top bar shows approximate distance in nt. Gray arrows represent the anti-sense ncRNAs IG200 (ncRv10250) and IG199 (ncRv10249), identified by Arnvig et al . ( 72 , 75 ). ( B ) ß-galactosidase assay of the individual promoter fusion constructs in M. bovis BCG wild type (black bar) and crp background (hashed bars). ( C ) Purified CRP Mt 's ability to bind to either site 1 or 2 in the upstream region of Rv0250c or sites 3 or 4 in the upstream region of Rv0249c. ( D ) Contribution of sites 2 and 3 to expression. Black bars, wild type, hashed bars, replaced promoter fusion construct at site 2 or site 3 as indicated by x-axis. ( E ) RT-PCR using cDNA generated in the absence (−) or presence (+) of reverse transcriptase. Heat killed genomic DNA from M. tuberculosis H37Rv (H). Corresponding products are indicated in panel (A). ( F ) Summary of data. Small arrows above sites 2 and 4 indicate the positions of the binding sites relative to the transcriptional start sites (tss) as published ( 71 ). Binding site 1 is centered 64 nt from the +1 of Rv0250c, while the center of binding site 3 is 67.5 nt from the +1 of Rv0249c. Sites 2 and 4 overlap tss's for Rv0250c and Rv0249c promoters, respectively. Small arrows under each locus indicate promoter fragments used in these studies. NT not tested. Shown are the means with standard deviations from three independent experiments. (*, P

    Techniques Used: β-Gal Assay, Construct, Purification, Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Binding Assay

    63) Product Images from "LINE-1 hypomethylation in normal colon mucosa is associated with poor survival in Chinese patients with sporadic colon cancer"

    Article Title: LINE-1 hypomethylation in normal colon mucosa is associated with poor survival in Chinese patients with sporadic colon cancer

    Journal: Oncotarget

    doi:

    Representative LMR results after pyrosequencing Bisulfite-treated DNA samples from adjacent normal mucosa were subjected to PCR amplification and were quantitatively analyzed by pyrosequencing. The C base marked in yellow served as a quality control of the bisulfite conversion efficiency. Four analyzed CpG sites are highlighted in blue, and the percent methylation rate is provided for each site. The mean percentage was computed as the LINE-1 methylation rate (LMR) for each case. Two cases with relatively higher (73.8%, A. ) or lower (19.4%, B. ) LMR were shown, respectively.
    Figure Legend Snippet: Representative LMR results after pyrosequencing Bisulfite-treated DNA samples from adjacent normal mucosa were subjected to PCR amplification and were quantitatively analyzed by pyrosequencing. The C base marked in yellow served as a quality control of the bisulfite conversion efficiency. Four analyzed CpG sites are highlighted in blue, and the percent methylation rate is provided for each site. The mean percentage was computed as the LINE-1 methylation rate (LMR) for each case. Two cases with relatively higher (73.8%, A. ) or lower (19.4%, B. ) LMR were shown, respectively.

    Techniques Used: Polymerase Chain Reaction, Amplification, Methylation

    Representative MSI status results after STR analysis Electropherograms of labeled PCR products targeting six microsatellite loci in paired tumor (upper) and normal (bottom) DNA samples from a representative patient: BAT26 and BAT25 (A) , D5S346 and D2S123 (B) , and BAT40 and D17S250 (C). The PCR product size is represented on the X-axis, and fluorescence units are represented on the Y-axis. For all the microsatellite loci, the tumor DNA sample showed altered allelic profiles compared to the matched normal DNA sample. Thus, this case was defined as MSI-H.
    Figure Legend Snippet: Representative MSI status results after STR analysis Electropherograms of labeled PCR products targeting six microsatellite loci in paired tumor (upper) and normal (bottom) DNA samples from a representative patient: BAT26 and BAT25 (A) , D5S346 and D2S123 (B) , and BAT40 and D17S250 (C). The PCR product size is represented on the X-axis, and fluorescence units are represented on the Y-axis. For all the microsatellite loci, the tumor DNA sample showed altered allelic profiles compared to the matched normal DNA sample. Thus, this case was defined as MSI-H.

    Techniques Used: Labeling, Polymerase Chain Reaction, Fluorescence

    64) Product Images from "MITF depletion elevates expression levels of ERBB3 receptor and its cognate ligand NRG1-beta in melanoma"

    Article Title: MITF depletion elevates expression levels of ERBB3 receptor and its cognate ligand NRG1-beta in melanoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10422

    MITF suppresses ERBB3 expression at the transcriptional level in various cell lines after siRNA transfections Assessment of mRNA and protein levels of MITF and ERBB3 in a panel of cell lines 72h after siRNA-induced reduction of MITF and ERBB3. A. Hermes 4C (immortalized melanocytes). B. SKMEL28 (BRAFV600E) C. MeWo (NF1) D. WM983B (BRAFV600E) E. WM1382 (wild-type for BRAF and NRAS). Graphs represent qRT-PCR expression data from three separate experiments normalized to untreated control cells and plotted as mean ± SD. * = p
    Figure Legend Snippet: MITF suppresses ERBB3 expression at the transcriptional level in various cell lines after siRNA transfections Assessment of mRNA and protein levels of MITF and ERBB3 in a panel of cell lines 72h after siRNA-induced reduction of MITF and ERBB3. A. Hermes 4C (immortalized melanocytes). B. SKMEL28 (BRAFV600E) C. MeWo (NF1) D. WM983B (BRAFV600E) E. WM1382 (wild-type for BRAF and NRAS). Graphs represent qRT-PCR expression data from three separate experiments normalized to untreated control cells and plotted as mean ± SD. * = p

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    MITF suppress the PI3K-pathway through NRG1-beta/ERBB3 signaling A-B. Representative western blots show the effect of MITF siRNA treatment on NRG1-beta/ERBB3 signaling pathway members, leading to p-AKT (S473) activation in WM983B (A) and MeWo (B) . Cell lines were transfected with MITF and ERBB3 siRNA alone and in combination for 72h, and treated either with or without (see Supplementary Figure S3A-S3B ) 10ng/ml NRG1-beta ligand 15min prior to harvesting. All experiments were performed in triplicate. Histone H3 was used as loading control. C-D. qRT-PCR data show mRNA elevation of NRG1-beta ligand after MITF depletion in WM983B (C) , WM1382 (D) and Hermes 4C (E). Graphs represent qRT-PCR expression data from three separate experiments normalized to untreated control cells and plotted as mean ± SD. * = p
    Figure Legend Snippet: MITF suppress the PI3K-pathway through NRG1-beta/ERBB3 signaling A-B. Representative western blots show the effect of MITF siRNA treatment on NRG1-beta/ERBB3 signaling pathway members, leading to p-AKT (S473) activation in WM983B (A) and MeWo (B) . Cell lines were transfected with MITF and ERBB3 siRNA alone and in combination for 72h, and treated either with or without (see Supplementary Figure S3A-S3B ) 10ng/ml NRG1-beta ligand 15min prior to harvesting. All experiments were performed in triplicate. Histone H3 was used as loading control. C-D. qRT-PCR data show mRNA elevation of NRG1-beta ligand after MITF depletion in WM983B (C) , WM1382 (D) and Hermes 4C (E). Graphs represent qRT-PCR expression data from three separate experiments normalized to untreated control cells and plotted as mean ± SD. * = p

    Techniques Used: Western Blot, Activation Assay, Transfection, Quantitative RT-PCR, Expressing

    Basal expression levels of MITF, ERBB3, SOX10 and FOXD3 in various melanoma cell lines A-D. qRT-PCR was used to evaluate mRNA levels of MITF (A) , ERBB3 (B) , SOX10 (C) , and FOXD3 (D) in melanoma cell lines by normalizing against immortalized cultured melanocytes (Hermes 4C). Bars represent mean ± SD of three separate experiments (E). Representative western blots of MITF, SOX10, FOXD3 and ERBB3 protein levels shown in 9 different cell lines representing various disease stage and genetic background. Histone H3 was used as loading control.
    Figure Legend Snippet: Basal expression levels of MITF, ERBB3, SOX10 and FOXD3 in various melanoma cell lines A-D. qRT-PCR was used to evaluate mRNA levels of MITF (A) , ERBB3 (B) , SOX10 (C) , and FOXD3 (D) in melanoma cell lines by normalizing against immortalized cultured melanocytes (Hermes 4C). Bars represent mean ± SD of three separate experiments (E). Representative western blots of MITF, SOX10, FOXD3 and ERBB3 protein levels shown in 9 different cell lines representing various disease stage and genetic background. Histone H3 was used as loading control.

    Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture, Western Blot

    65) Product Images from "Pharmacological modulation of the AKT/microRNA-199a-5p/CAV1 pathway ameliorates cystic fibrosis lung hyper-inflammation"

    Article Title: Pharmacological modulation of the AKT/microRNA-199a-5p/CAV1 pathway ameliorates cystic fibrosis lung hyper-inflammation

    Journal: Nature communications

    doi: 10.1038/ncomms7221

    Celecoxib rescues the miR-199a-5p/CAV1 pathways by stimulating PI3K-AKT signaling in CF MΦs, and decreases the lung hyper-inflammatory response to LPS in CF-affected mice ( A ) qPCR for IL-6 (left), miR-199a-5p (middle) and CAV1(right) and ( B) WB and densitometric analysis for HO-1 in CF murine MΦs untreated or treated with LPS, in absence or presence of celecoxib; (C) qPCR for miR-199a-5p (left) and CAV1 (right) in WT and CF murine MΦs treated with LPS, in presence or absence of celecoxib and of PI3K-AKT inhibitor LY94002; (D) WB and densitometric analysis for AKT, pAKT and B-actin in WT and CF murine MΦs treated with LPS for 4h in presence or absence of celecoxib; (E) In vivo experiment schematic representation; total and differential BAL fluid cell number (F) , hematoxylin/eosin staining in paraffin embedded lung tissues ( G ), qPCR for IL-6, miR-199a-5p and CAV1 (H) and body weigh loss (I) from WT and CF mice treated chronically (10 days) with celecoxib (Celebrex) and then challenged with LPS for three days. Mice were sacrificed 24h after last nebulization. For qPCR, miR-199a levels are normalized to RNU6B and CAV1 and IL-6 expression to S18. For WB, protein fold increase is normalized to B-actin or total AKT (pAKT). For the in vitro experiments, the data are the result of three experimental biological repeats; for the in vivo study, three mice were used for each group, and the experiment was repeated independently twice (total: 6 mice per group). Statistical analyses were conducted using one-sided two-sample t-tests (q-PCR) or two-sample unequal variance t-tests (BAL fluid cell numbers). Error bars indicate standard deviation. The symbol * indicates a statistically significant difference among groups with a P values
    Figure Legend Snippet: Celecoxib rescues the miR-199a-5p/CAV1 pathways by stimulating PI3K-AKT signaling in CF MΦs, and decreases the lung hyper-inflammatory response to LPS in CF-affected mice ( A ) qPCR for IL-6 (left), miR-199a-5p (middle) and CAV1(right) and ( B) WB and densitometric analysis for HO-1 in CF murine MΦs untreated or treated with LPS, in absence or presence of celecoxib; (C) qPCR for miR-199a-5p (left) and CAV1 (right) in WT and CF murine MΦs treated with LPS, in presence or absence of celecoxib and of PI3K-AKT inhibitor LY94002; (D) WB and densitometric analysis for AKT, pAKT and B-actin in WT and CF murine MΦs treated with LPS for 4h in presence or absence of celecoxib; (E) In vivo experiment schematic representation; total and differential BAL fluid cell number (F) , hematoxylin/eosin staining in paraffin embedded lung tissues ( G ), qPCR for IL-6, miR-199a-5p and CAV1 (H) and body weigh loss (I) from WT and CF mice treated chronically (10 days) with celecoxib (Celebrex) and then challenged with LPS for three days. Mice were sacrificed 24h after last nebulization. For qPCR, miR-199a levels are normalized to RNU6B and CAV1 and IL-6 expression to S18. For WB, protein fold increase is normalized to B-actin or total AKT (pAKT). For the in vitro experiments, the data are the result of three experimental biological repeats; for the in vivo study, three mice were used for each group, and the experiment was repeated independently twice (total: 6 mice per group). Statistical analyses were conducted using one-sided two-sample t-tests (q-PCR) or two-sample unequal variance t-tests (BAL fluid cell numbers). Error bars indicate standard deviation. The symbol * indicates a statistically significant difference among groups with a P values

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot, In Vivo, Staining, Expressing, In Vitro, Polymerase Chain Reaction, Standard Deviation

    66) Product Images from "Editing of Genomic TNFSF9 by CRISPR-Cas9 Can Be Followed by Re-Editing of Its Transcript"

    Article Title: Editing of Genomic TNFSF9 by CRISPR-Cas9 Can Be Followed by Re-Editing of Its Transcript

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2018.0209

    cDNA sequence of mRNA de novo transcribed from TNFSF9 in HepG2 clone 9-1 (A) HepG2 clone 9-1 cells were incubated with 500 ng/ml LPS for the indicated times. cDNA was synthesized from total RNA, and TNFSF9 expression was quantified by real-time PCR as described in “Materials and Methods”. (B) HepG2 cells were incubated with 500 ng/ml LPS in the presence of 5-ethynyl uridine (EU) for 16 hrs. EU-labeled RNA was isolated as described in “Materials and Methods”. TNFSF9 cDNA was amplified by PCR and the PCR products were isolated and sequenced. This experiment is representative of four independent experiments.
    Figure Legend Snippet: cDNA sequence of mRNA de novo transcribed from TNFSF9 in HepG2 clone 9-1 (A) HepG2 clone 9-1 cells were incubated with 500 ng/ml LPS for the indicated times. cDNA was synthesized from total RNA, and TNFSF9 expression was quantified by real-time PCR as described in “Materials and Methods”. (B) HepG2 cells were incubated with 500 ng/ml LPS in the presence of 5-ethynyl uridine (EU) for 16 hrs. EU-labeled RNA was isolated as described in “Materials and Methods”. TNFSF9 cDNA was amplified by PCR and the PCR products were isolated and sequenced. This experiment is representative of four independent experiments.

    Techniques Used: Sequencing, Incubation, Synthesized, Expressing, Real-time Polymerase Chain Reaction, Labeling, Isolation, Amplification, Polymerase Chain Reaction

    DNA sequence of genomic TNFSF9 gene edited by CRISPR-Cas9 The TNFSF9 genes in genomic DNAs from wild type and mutated HepG2 cells were amplified by PCR. The PCR products were isolated and sequenced as described in “Materials and Methods”. Left panels are chromatograms of the genomic TNFSF9 sequence. On the right are shown the DNA sequences around the region of the mutated triplet in the wildtype and mutant clones. This is one representative of five independent experiments.
    Figure Legend Snippet: DNA sequence of genomic TNFSF9 gene edited by CRISPR-Cas9 The TNFSF9 genes in genomic DNAs from wild type and mutated HepG2 cells were amplified by PCR. The PCR products were isolated and sequenced as described in “Materials and Methods”. Left panels are chromatograms of the genomic TNFSF9 sequence. On the right are shown the DNA sequences around the region of the mutated triplet in the wildtype and mutant clones. This is one representative of five independent experiments.

    Techniques Used: Sequencing, CRISPR, Amplification, Polymerase Chain Reaction, Isolation, Mutagenesis, Clone Assay

    Heteroduplex formation by the cDNA of de novo transcripts of TNFSF9 from HepG2 clone 9-1 Total RNA and EU-labeled RNAs were isolated from clone 9-1 HepG2 cells. cDNA was synthesized and the TNFSF9 cDNA was amplified by PCR as described in “Materials and Methods”. The PCR products were annealed and incubated in the presence or absence of T7E1 endonuclease, and the products were fractionated on 1.2% agarose gels and visualized under UV. This is one representative of three independent experiments.
    Figure Legend Snippet: Heteroduplex formation by the cDNA of de novo transcripts of TNFSF9 from HepG2 clone 9-1 Total RNA and EU-labeled RNAs were isolated from clone 9-1 HepG2 cells. cDNA was synthesized and the TNFSF9 cDNA was amplified by PCR as described in “Materials and Methods”. The PCR products were annealed and incubated in the presence or absence of T7E1 endonuclease, and the products were fractionated on 1.2% agarose gels and visualized under UV. This is one representative of three independent experiments.

    Techniques Used: Labeling, Isolation, Synthesized, Amplification, Polymerase Chain Reaction, Incubation

    cDNA sequence of mRNA transcribed from mutant genomic TNFSF9 TNFSF9 cDNA was synthesized and amplified by PCR from total RNA extracted from wild type and mutated HepG2 cells. The PCR products were isolated and sequenced as described in “Materials and Methods”. Left panels are chromatograms of the TNFSF9 cDNA sequences. On the right are shown the cDNA sequences around the region of the mutated triplet in the wild type and mutant clones. This is one representative of four independent experiments.
    Figure Legend Snippet: cDNA sequence of mRNA transcribed from mutant genomic TNFSF9 TNFSF9 cDNA was synthesized and amplified by PCR from total RNA extracted from wild type and mutated HepG2 cells. The PCR products were isolated and sequenced as described in “Materials and Methods”. Left panels are chromatograms of the TNFSF9 cDNA sequences. On the right are shown the cDNA sequences around the region of the mutated triplet in the wild type and mutant clones. This is one representative of four independent experiments.

    Techniques Used: Sequencing, Mutagenesis, Synthesized, Amplification, Polymerase Chain Reaction, Isolation, Clone Assay

    67) Product Images from "Development of Genetic System to Inactivate a Borrelia turicatae Surface Protein Selectively Produced within the Salivary Glands of the Arthropod Vector"

    Article Title: Development of Genetic System to Inactivate a Borrelia turicatae Surface Protein Selectively Produced within the Salivary Glands of the Arthropod Vector

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002514

    Constructing a deletion vector for brpA. brpA was amplified including 1,000 nucleotides up and down stream of the gene (A) and cloned into the TopoXL vector, constructing Topo-XL:: brpA (B). brpA was removed from the vector by PCR amplification adding AvrII and NheI restriction sites (C and D) and the amplicon was double digested. The B. turicatae flgB P -gent was amplified adding AvrII and SpeI restriction sites, double digested, and the knockout vector constructed by ligation (E).
    Figure Legend Snippet: Constructing a deletion vector for brpA. brpA was amplified including 1,000 nucleotides up and down stream of the gene (A) and cloned into the TopoXL vector, constructing Topo-XL:: brpA (B). brpA was removed from the vector by PCR amplification adding AvrII and NheI restriction sites (C and D) and the amplicon was double digested. The B. turicatae flgB P -gent was amplified adding AvrII and SpeI restriction sites, double digested, and the knockout vector constructed by ligation (E).

    Techniques Used: Plasmid Preparation, Amplification, Clone Assay, Polymerase Chain Reaction, Knock-Out, Construct, Ligation

    68) Product Images from "Androgen Signaling Disruption during Fetal and Postnatal Development Affects Androgen Receptor and Connexin 43 Expression and Distribution in Adult Boar Prostate"

    Article Title: Androgen Signaling Disruption during Fetal and Postnatal Development Affects Androgen Receptor and Connexin 43 Expression and Distribution in Adult Boar Prostate

    Journal: BioMed Research International

    doi: 10.1155/2013/407678

    Androgen receptor and Cx43 mRNAs and protein expression in prostates of control and flutamide-exposed boars. ((a), (c)) Androgen receptor and Cx43 mRNAs expression. As an intrinsic control, the GAPDH mRNA level was measured in the samples. Representative gels electrophoresis of qualitative expression of AR (a), Cx43 (c) and GAPDH mRNAs. Line N1—negative control without cDNA template, line N2—negative control without reverse transcribed RNA. Relative expression of mRNA for AR (a) and Cx43 (c) determined using real-time RT-PCR analysis. Relative quantification (RQ) is expressed as means ± SD. Significant differences from control values are denoted as * P
    Figure Legend Snippet: Androgen receptor and Cx43 mRNAs and protein expression in prostates of control and flutamide-exposed boars. ((a), (c)) Androgen receptor and Cx43 mRNAs expression. As an intrinsic control, the GAPDH mRNA level was measured in the samples. Representative gels electrophoresis of qualitative expression of AR (a), Cx43 (c) and GAPDH mRNAs. Line N1—negative control without cDNA template, line N2—negative control without reverse transcribed RNA. Relative expression of mRNA for AR (a) and Cx43 (c) determined using real-time RT-PCR analysis. Relative quantification (RQ) is expressed as means ± SD. Significant differences from control values are denoted as * P

    Techniques Used: Expressing, Electrophoresis, Negative Control, Quantitative RT-PCR

    69) Product Images from "Two Common Bean Genotypes with Contrasting Response to Phosphorus Deficiency Show Variations in the microRNA 399-Mediated PvPHO2 Regulation within the PvPHR1 Signaling Pathway"

    Article Title: Two Common Bean Genotypes with Contrasting Response to Phosphorus Deficiency Show Variations in the microRNA 399-Mediated PvPHO2 Regulation within the PvPHR1 Signaling Pathway

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms14048328

    microRNA-directed cleavage of PvPHO2 mRNA. 5′RACE analysis was performed for PvPHO2 using oligonucleotides to specifically determine cleavage directed by PvmiR399 at two recognition sites (1 and 3) in the BAT477 and DOR364 genotypes. ( A ) PCR products corresponding to cleaved PvPHO2 fragments from BAT477 variety were amplified by nested PCR and resolved in a 6% PA gel (arrows indicate size and position of predicted fragments; for procedure details, see the Experimental Section). The corresponding PCR fragments from BAT477 were cloned and sequenced to confirm their identity; ( B ) The alignment between PvPHO2 binding sites 1 and 3 and PvmiR399b is shown; arrows indicate site of cleavage recovered, and numbers refer to number of independent clones analyzed. C . PCR fragments corresponding to cleavage of NAC1 by miR164 that is unrelated to the P -deficiency response.
    Figure Legend Snippet: microRNA-directed cleavage of PvPHO2 mRNA. 5′RACE analysis was performed for PvPHO2 using oligonucleotides to specifically determine cleavage directed by PvmiR399 at two recognition sites (1 and 3) in the BAT477 and DOR364 genotypes. ( A ) PCR products corresponding to cleaved PvPHO2 fragments from BAT477 variety were amplified by nested PCR and resolved in a 6% PA gel (arrows indicate size and position of predicted fragments; for procedure details, see the Experimental Section). The corresponding PCR fragments from BAT477 were cloned and sequenced to confirm their identity; ( B ) The alignment between PvPHO2 binding sites 1 and 3 and PvmiR399b is shown; arrows indicate site of cleavage recovered, and numbers refer to number of independent clones analyzed. C . PCR fragments corresponding to cleavage of NAC1 by miR164 that is unrelated to the P -deficiency response.

    Techniques Used: Polymerase Chain Reaction, Amplification, Nested PCR, Clone Assay, Binding Assay

    Expression of regulatory genes from the PvPHR1 signal pathway in roots of common bean BAT477 (solid bars) and DOR364 (hatched bars) genotypes. Plants were grown for three weeks under P sufficient (blue bars) or in P deficient (orange bars) conditions. Transcript levels were determined by qRT-PCR. Values are the mean (±SE) from three independent experiments with nine replicates per experiment. * Significantly different response to P deficiency between BAT477 and DOR364 ( p ≤ 0.05).
    Figure Legend Snippet: Expression of regulatory genes from the PvPHR1 signal pathway in roots of common bean BAT477 (solid bars) and DOR364 (hatched bars) genotypes. Plants were grown for three weeks under P sufficient (blue bars) or in P deficient (orange bars) conditions. Transcript levels were determined by qRT-PCR. Values are the mean (±SE) from three independent experiments with nine replicates per experiment. * Significantly different response to P deficiency between BAT477 and DOR364 ( p ≤ 0.05).

    Techniques Used: Expressing, Quantitative RT-PCR

    70) Product Images from "The Immunomodulation of Acetylcholinesterase in Zhikong Scallop Chlamys farreri"

    Article Title: The Immunomodulation of Acetylcholinesterase in Zhikong Scallop Chlamys farreri

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030828

    Tissue distribution of the CfAChE transcripts detected by SYBR Green RT-PCR. CfAChE transcript level in adductor muscle, mantle, gill, hepatopancreas, kidney and gonad of six adult scallops was normalized to that of haemocytes. Vertical bars represented the mean ± S.E. (N = 6), and bars with * are significantly different ( P
    Figure Legend Snippet: Tissue distribution of the CfAChE transcripts detected by SYBR Green RT-PCR. CfAChE transcript level in adductor muscle, mantle, gill, hepatopancreas, kidney and gonad of six adult scallops was normalized to that of haemocytes. Vertical bars represented the mean ± S.E. (N = 6), and bars with * are significantly different ( P

    Techniques Used: SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction

    71) Product Images from "The Pyrimidine Nucleotide Biosynthetic Pathway Modulates Production of Biofilm Determinants in Escherichia coli"

    Article Title: The Pyrimidine Nucleotide Biosynthetic Pathway Modulates Production of Biofilm Determinants in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031252

    Congo red binding by E. coli strains deficient in pyrimidine sensing ( cytR and rutR mutants) and purine biosynthesis ( purH mutant). 4A. The MG1655 strain and its isogenic mutants in the purH , cytR and rutR genes were spotted either on CR medium (left panel) or on CR(ura) medium (right panel) and grown for 24 hours at 30°C. Plates were incubated for 48 hours at 4°C to enhance Congo red binding. Determination of transcript levels. 4B. Relative expression of either the csgD gene (left panel) or the udp gene (right panel) was determined by Real-Time PCR on RNA extracted from overnight cultures of MG1655 and of its isogenic purH and cytR mutants. 16S RNA transcript was used as reference gene. Δ Ct values between the genes of interest and 16S RNA were set at 1 for MG1655 in LB1/4 medium, and transcript levels in other strains and/or growth conditions are expressed as relative values. Experiments were repeated at least three times, each time in duplicate; standard deviations were always lower than 5%.
    Figure Legend Snippet: Congo red binding by E. coli strains deficient in pyrimidine sensing ( cytR and rutR mutants) and purine biosynthesis ( purH mutant). 4A. The MG1655 strain and its isogenic mutants in the purH , cytR and rutR genes were spotted either on CR medium (left panel) or on CR(ura) medium (right panel) and grown for 24 hours at 30°C. Plates were incubated for 48 hours at 4°C to enhance Congo red binding. Determination of transcript levels. 4B. Relative expression of either the csgD gene (left panel) or the udp gene (right panel) was determined by Real-Time PCR on RNA extracted from overnight cultures of MG1655 and of its isogenic purH and cytR mutants. 16S RNA transcript was used as reference gene. Δ Ct values between the genes of interest and 16S RNA were set at 1 for MG1655 in LB1/4 medium, and transcript levels in other strains and/or growth conditions are expressed as relative values. Experiments were repeated at least three times, each time in duplicate; standard deviations were always lower than 5%.

    Techniques Used: Binding Assay, Mutagenesis, Incubation, Expressing, Real-time Polymerase Chain Reaction

    72) Product Images from "pitx2 Deficiency Results in Abnormal Ocular and Craniofacial Development in Zebrafish"

    Article Title: pitx2 Deficiency Results in Abnormal Ocular and Craniofacial Development in Zebrafish

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030896

    zebrafish pitx2 knockdown and associated phenotype. A . Schematic drawing of pitx2 genomic structure. Exons are shown as numbered boxes, sizes are indicated at the top (for exons) or at the bottom (for introns). The positions of primers to amplify pitx2 transcripts are shown and numbered 1–3; primers 1 and 3 are used for pitx2a and 2 and 3 for pitx2c . The position of antisense morpholino oligonucleotides, pitx2a ATG , pitx2c ATG and pitx2 ex4/5sp , are shown with red lines. B . RT-PCR of pitx2 expression in pitx2 ex4/5 morphants. Please note a complete absence of normal pitx2 transcripts (indicated with black arrows) and the presence of an abnormal large PCR product (indicated with red arrowheads) in mRNA extracted from pitx2 ex4/5 embryos at 24–48-hpf, the presence of both normal (diminished) and abnormal products at 72–96-hpf pitx2 ex4/5 , and normal levels of pitx2 by 120-hpf due to weakening of morpholino effects. C . DNA sequencing of the abnormal PCR product observed in pitx2 ex4/5 morphant embryos identified the presence of the 902-bp intron 4 in the pitx2 ex4/5 transcript consistent with aberrant splicing (forward sequence is shown and the beginning of the intron is indicated with a red arrow; exon 4 sequence is shown in upper case while intron 4 is in lower case letters; the exon-intron junction sequence corresponding to the pitx2 ex4/5 antisense oligomer is indicated in red). Therefore, the pitx2 ex4/5 protein is predicted to contain partial pitx2 sequence (lacking amino acids encoded by exon 5) followed by 10 erroneous amino acids ( pitx2 ex4/5 stop codon is indicated with red box). D . Representative images of pitx2 ex4/5 morphants, control morpholino-injected embryos and larvae developed from uninjected eggs at 48-hpf, 96-hpf and 7-dpf. E . Bar graph showing the distribution of observed embryonic phenotypes following pitx2 ex4/5 morpholino injections into p53 −/− and wild-type zebrafish eggs. e- eye, j- jaw, pe- pericardial edema.
    Figure Legend Snippet: zebrafish pitx2 knockdown and associated phenotype. A . Schematic drawing of pitx2 genomic structure. Exons are shown as numbered boxes, sizes are indicated at the top (for exons) or at the bottom (for introns). The positions of primers to amplify pitx2 transcripts are shown and numbered 1–3; primers 1 and 3 are used for pitx2a and 2 and 3 for pitx2c . The position of antisense morpholino oligonucleotides, pitx2a ATG , pitx2c ATG and pitx2 ex4/5sp , are shown with red lines. B . RT-PCR of pitx2 expression in pitx2 ex4/5 morphants. Please note a complete absence of normal pitx2 transcripts (indicated with black arrows) and the presence of an abnormal large PCR product (indicated with red arrowheads) in mRNA extracted from pitx2 ex4/5 embryos at 24–48-hpf, the presence of both normal (diminished) and abnormal products at 72–96-hpf pitx2 ex4/5 , and normal levels of pitx2 by 120-hpf due to weakening of morpholino effects. C . DNA sequencing of the abnormal PCR product observed in pitx2 ex4/5 morphant embryos identified the presence of the 902-bp intron 4 in the pitx2 ex4/5 transcript consistent with aberrant splicing (forward sequence is shown and the beginning of the intron is indicated with a red arrow; exon 4 sequence is shown in upper case while intron 4 is in lower case letters; the exon-intron junction sequence corresponding to the pitx2 ex4/5 antisense oligomer is indicated in red). Therefore, the pitx2 ex4/5 protein is predicted to contain partial pitx2 sequence (lacking amino acids encoded by exon 5) followed by 10 erroneous amino acids ( pitx2 ex4/5 stop codon is indicated with red box). D . Representative images of pitx2 ex4/5 morphants, control morpholino-injected embryos and larvae developed from uninjected eggs at 48-hpf, 96-hpf and 7-dpf. E . Bar graph showing the distribution of observed embryonic phenotypes following pitx2 ex4/5 morpholino injections into p53 −/− and wild-type zebrafish eggs. e- eye, j- jaw, pe- pericardial edema.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, DNA Sequencing, Sequencing, Injection

    73) Product Images from "Molecular and Biochemical Effects of a Kola Nut Extract on Androgen Receptor-Mediated Pathways"

    Article Title: Molecular and Biochemical Effects of a Kola Nut Extract on Androgen Receptor-Mediated Pathways

    Journal: Journal of Toxicology

    doi: 10.1155/2009/530279

    Stimulation of PSA mRNA expression by Biz-2. LNCaP cells were induced with Biz-2 or DHT, and 5 μ g of DNase-I-treated RNA isolated from induced or uninduced cells were subjected to two-step Taqman real-time RT-PCR (see Section 2 ). The relative expression of PSA mRNA expression was calculated by the 2 ΔΔ C t method. First, relative quantitation of PSA mRNA expression was performed by first normalizing the C t values of PSA amplification against the C t values of endogenous 18S rRNA, then the resulting C t values were normalized using the C t value of the vehicle control sample. (a) Relative amplification plot of mRNA expression (1–NTC. 2–18S rRNA at 24 hours, 3 and 4- CON at 6 and 24 hours, 5 and 6- Biz-2 at 6 and 24 hours). (b) PSA mRNA expression relative to time. (c) Dose-dependent response of PSA to Biz-2. The values are mean ± SEM of induction samples analyzed in triplicate.
    Figure Legend Snippet: Stimulation of PSA mRNA expression by Biz-2. LNCaP cells were induced with Biz-2 or DHT, and 5 μ g of DNase-I-treated RNA isolated from induced or uninduced cells were subjected to two-step Taqman real-time RT-PCR (see Section 2 ). The relative expression of PSA mRNA expression was calculated by the 2 ΔΔ C t method. First, relative quantitation of PSA mRNA expression was performed by first normalizing the C t values of PSA amplification against the C t values of endogenous 18S rRNA, then the resulting C t values were normalized using the C t value of the vehicle control sample. (a) Relative amplification plot of mRNA expression (1–NTC. 2–18S rRNA at 24 hours, 3 and 4- CON at 6 and 24 hours, 5 and 6- Biz-2 at 6 and 24 hours). (b) PSA mRNA expression relative to time. (c) Dose-dependent response of PSA to Biz-2. The values are mean ± SEM of induction samples analyzed in triplicate.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Quantitation Assay, Amplification

    Flutamide inhibition of Biz-2 stimulation of PSA mRNA expression. LNCaP cells were induced with Biz-2 in the presence or absence of flutamide, and RNA from induced or uninduced cells was subjected to two-step Taqman real-time RT-PCR. Relative quantitation of PSA mRNA expression was preformed as described in Figure 2 . CON: vehicle control; FLU: flutamide; Biz-2: Bizzy nut extract-2; RES: resveratrol; Act.D: actinomycin-D; CHX: cycloheximide. The values are the mean ± SEM of three separate experiments performed in triplicate.
    Figure Legend Snippet: Flutamide inhibition of Biz-2 stimulation of PSA mRNA expression. LNCaP cells were induced with Biz-2 in the presence or absence of flutamide, and RNA from induced or uninduced cells was subjected to two-step Taqman real-time RT-PCR. Relative quantitation of PSA mRNA expression was preformed as described in Figure 2 . CON: vehicle control; FLU: flutamide; Biz-2: Bizzy nut extract-2; RES: resveratrol; Act.D: actinomycin-D; CHX: cycloheximide. The values are the mean ± SEM of three separate experiments performed in triplicate.

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Quantitation Assay, Activated Clotting Time Assay

    74) Product Images from "Comprehensive circRNA expression profile during ischemic postconditioning attenuating hepatic ischemia/reperfusion injury"

    Article Title: Comprehensive circRNA expression profile during ischemic postconditioning attenuating hepatic ischemia/reperfusion injury

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36443-8

    Validation of selected circRNAs, miRNAs and mRNAs by qRT-PCR. ( a ) 6 circRNAs were significantly amplified by qRT-PCR and consistent with the microarray results. (b) 9 miRNAs were significantly amplified by qRT-PCR and were inversely correlated with their corresponding circRNAs. (c) 6 mRNAs were significantly amplified by qRT-PCR while only Epha2, Arhgap32, Heatr1 and Egr1 had an identical expression trend with their corresponding circRNAs. * p
    Figure Legend Snippet: Validation of selected circRNAs, miRNAs and mRNAs by qRT-PCR. ( a ) 6 circRNAs were significantly amplified by qRT-PCR and consistent with the microarray results. (b) 9 miRNAs were significantly amplified by qRT-PCR and were inversely correlated with their corresponding circRNAs. (c) 6 mRNAs were significantly amplified by qRT-PCR while only Epha2, Arhgap32, Heatr1 and Egr1 had an identical expression trend with their corresponding circRNAs. * p

    Techniques Used: Quantitative RT-PCR, Amplification, Microarray, Expressing

    Effect of mmu_circRNA_005186 on LPS-induced inflammation in RAW264.7 cells. (a , b ) ELISA assay demonstrated that the supernatant concentrations of TNF-α and IL-1β released from RAW 264.7 cells were significantly lower in the si-005186 group than the negative control. (c) qRT-PCR showed that miR-124-3p was upregulated by 6-fold in the si-005186 group. (d) mmu_circRNA_005186 silencing inhibited the expression level of Epha2 in LPS-induced inflammation. * p
    Figure Legend Snippet: Effect of mmu_circRNA_005186 on LPS-induced inflammation in RAW264.7 cells. (a , b ) ELISA assay demonstrated that the supernatant concentrations of TNF-α and IL-1β released from RAW 264.7 cells were significantly lower in the si-005186 group than the negative control. (c) qRT-PCR showed that miR-124-3p was upregulated by 6-fold in the si-005186 group. (d) mmu_circRNA_005186 silencing inhibited the expression level of Epha2 in LPS-induced inflammation. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Negative Control, Quantitative RT-PCR, Expressing

    75) Product Images from "Skeletonema marinoi as a new genetic model for marine chain-forming diatoms"

    Article Title: Skeletonema marinoi as a new genetic model for marine chain-forming diatoms

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41085-5

    Mapping the genomic insertion sites in several representative transformants using TAIL-PCR. TAIL-PCR was performed on genomic DNA extracted from the transformants SM1–2, SM10, SM12–13 and SM15 using one of the SAD1–4 degenerate primers in each reaction in combination with the construct-specific primers for either the promoter or terminator ends. The final PCR products were separated and visualized by gel electrophoresis as shown, with selected individual products then purified and sequenced.
    Figure Legend Snippet: Mapping the genomic insertion sites in several representative transformants using TAIL-PCR. TAIL-PCR was performed on genomic DNA extracted from the transformants SM1–2, SM10, SM12–13 and SM15 using one of the SAD1–4 degenerate primers in each reaction in combination with the construct-specific primers for either the promoter or terminator ends. The final PCR products were separated and visualized by gel electrophoresis as shown, with selected individual products then purified and sequenced.

    Techniques Used: Polymerase Chain Reaction, Construct, Nucleic Acid Electrophoresis, Purification

    Genetic transformation of S . marinoi . ( A ) Schematic designs of the linear DNA constructs for the transformation of S . marinoi R05AC. Two different promoter/terminator regions were chosen to express the selectable marker gene ( bleo R ) conferring resistance to the antibiotic zeocin; fcpB , gene for the P . tricornutum fucoxanthin-chlorophyll protein; lsu4e , gene for the S . marinoi 80 S ribosomal large subunit protein 4e. Also shown are the two oligonucleotides (Bleo1 and –2) used to PCR amplify the bleo R gene. ( B ) Sensitivity of S . marinoi to the antibiotic zeocin relative to that of P . tricornutum . Growth was monitored daily by chlorophyll fluorescence for one week in the presence of different zeocin concentrations. Shown are averages and standard deviation of four replicate cultures. ( C ) Amplification of the bleo R gene with Bleo1 and Bleo2 primers in representative transformants using the fcpB:bleoR or lsu4e:bleoR constructs.
    Figure Legend Snippet: Genetic transformation of S . marinoi . ( A ) Schematic designs of the linear DNA constructs for the transformation of S . marinoi R05AC. Two different promoter/terminator regions were chosen to express the selectable marker gene ( bleo R ) conferring resistance to the antibiotic zeocin; fcpB , gene for the P . tricornutum fucoxanthin-chlorophyll protein; lsu4e , gene for the S . marinoi 80 S ribosomal large subunit protein 4e. Also shown are the two oligonucleotides (Bleo1 and –2) used to PCR amplify the bleo R gene. ( B ) Sensitivity of S . marinoi to the antibiotic zeocin relative to that of P . tricornutum . Growth was monitored daily by chlorophyll fluorescence for one week in the presence of different zeocin concentrations. Shown are averages and standard deviation of four replicate cultures. ( C ) Amplification of the bleo R gene with Bleo1 and Bleo2 primers in representative transformants using the fcpB:bleoR or lsu4e:bleoR constructs.

    Techniques Used: Transformation Assay, Construct, Marker, Polymerase Chain Reaction, Fluorescence, Standard Deviation, Amplification

    Schematic representation of the genomic insertion site in selected S . marinoi transformants. Shown on the left are the predicted gene models disrupted by the insertion of the transformed DNA construct (blue triangle) in ten transformants (SM1–2, SM4, SM7, SM10–13 and SM15–16), with the green regions representing exons. The shown genomic regions correspond to at least one mRNA identified by RNA sequencing, the 5′ end of which is indicated by the black arrow. Underneath each gene map is shown a scale corresponding to 500 bp. Also indicated for each transformant are the primers specific for regions flanking the inserted construct used to determine the segregation state as shown on the right, confirming either heterozygosity or homozygosity for the mutation. PCR was performed on genomic DNA extracted from wild type S . marinoi and the indicated transformant. The amplified products were separated and visualized by gel electrophoresis. The identity of all PCR products was confirmed by DNA sequencing, with non-specific products denoted with an asterisk.
    Figure Legend Snippet: Schematic representation of the genomic insertion site in selected S . marinoi transformants. Shown on the left are the predicted gene models disrupted by the insertion of the transformed DNA construct (blue triangle) in ten transformants (SM1–2, SM4, SM7, SM10–13 and SM15–16), with the green regions representing exons. The shown genomic regions correspond to at least one mRNA identified by RNA sequencing, the 5′ end of which is indicated by the black arrow. Underneath each gene map is shown a scale corresponding to 500 bp. Also indicated for each transformant are the primers specific for regions flanking the inserted construct used to determine the segregation state as shown on the right, confirming either heterozygosity or homozygosity for the mutation. PCR was performed on genomic DNA extracted from wild type S . marinoi and the indicated transformant. The amplified products were separated and visualized by gel electrophoresis. The identity of all PCR products was confirmed by DNA sequencing, with non-specific products denoted with an asterisk.

    Techniques Used: Transformation Assay, Construct, RNA Sequencing Assay, Mutagenesis, Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis, DNA Sequencing

    Modified TAIL-PCR approach used to map the genomic insertion sites in S . marinoi transformants. ( A ) Schematic representation of the two sets of nested oligonucleotides specific for the bleo R gene to map the genomic flanking regions at either the promoter (Bleo3, –5, –6) or terminator (Bleo4, -7, -8) end using TAIL-PCR. Each set of nested primers are combined with one of four degenerate primers (SAD1-4) that anneal at a high frequency within the genome. ( B ) Schematic representation of the TAIL-PCR method used to identify the insertion site within an arbitrary gene (gene X) from the promoter end of the construct. In the first round of PCR amplification (1° reaction), the SAD primers produce numerous non-specific products in addition to the target fragment. In the following rounds (2° and 3°), the SAD primer is replaced with the specific SSMP primer, along with additional construct-specific primers (Bleo5 and -3) to concentrate the target fragment. The final products are size separated by gel electrophoresis, with each purified for DNA sequencing. Sequences are then matched against the construct sequence and the R05AC reference genome to identify the insertion site.
    Figure Legend Snippet: Modified TAIL-PCR approach used to map the genomic insertion sites in S . marinoi transformants. ( A ) Schematic representation of the two sets of nested oligonucleotides specific for the bleo R gene to map the genomic flanking regions at either the promoter (Bleo3, –5, –6) or terminator (Bleo4, -7, -8) end using TAIL-PCR. Each set of nested primers are combined with one of four degenerate primers (SAD1-4) that anneal at a high frequency within the genome. ( B ) Schematic representation of the TAIL-PCR method used to identify the insertion site within an arbitrary gene (gene X) from the promoter end of the construct. In the first round of PCR amplification (1° reaction), the SAD primers produce numerous non-specific products in addition to the target fragment. In the following rounds (2° and 3°), the SAD primer is replaced with the specific SSMP primer, along with additional construct-specific primers (Bleo5 and -3) to concentrate the target fragment. The final products are size separated by gel electrophoresis, with each purified for DNA sequencing. Sequences are then matched against the construct sequence and the R05AC reference genome to identify the insertion site.

    Techniques Used: Modification, Polymerase Chain Reaction, Construct, Amplification, Nucleic Acid Electrophoresis, Purification, DNA Sequencing, Sequencing

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    Whole Genome Amplification:

    Article Title: Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR(R) Yfiler(R) PCR amplification kit
    Article Snippet: Because of very low DNA quantities available for the Leipzig samples, a whole genome amplification procedure was performed before Yfiler PCR analysis using the GenomiPhi DNA Amplification Kit (GE Healthcare, Little Chalfont, UK). .. The Y-STRs included in the AmpFl STR® Yfiler® PCR amplification kit (Applied Biosystems, Inc.): DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS348, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4 were genotyped according to the instructions provided by the manufacturer and using a gold-plated silver block GeneAmp®PCR System 9700 (Applied Biosystems, Inc.).

    Positive Control:

    Article Title: Population Genetic Analysis of Haplotypes Based on 17 Short Tandem Repeat Loci on Y Chromosome in Population Sample from Eastern Croatia
    Article Snippet: After isolation, the genomic DNA content of each sample was determined by quantitative real-time polymerase chain reaction (PCR) using the Quantifiler™ Human Male DNA Quantification kit (Applied Biosystems, Foster City, CA, USA), which includes an internal positive control to test for the presence of PCR inhibitors in the DNA extracts. .. Genomic DNA (1 ng) was amplified using the AmpFISTR Yfiler PCR amplification kit (Applied Biosystems), which coamplifies 17 Y-STRs: DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and GATAH4.

    Polymerase Chain Reaction:

    Article Title: The genetic evidence for human origin of Jivaroan shrunken heads in collections from the Polish museums
    Article Snippet: .. Five commercially available reagent kits for typing of genomic short tandem repeat (STR) markers were used to study nuclear DNA (nDNA): AmpFlSTR Identifiler PCR Amplification Kit, GlobalFiler Amplification Kit, AmpFlSTR YFiler PCR Amplification Kit, and AmpFlSTR YFiler Plus PCR Amplification Kit (all from Applied Biosystems, Foster City, USA). .. Investigator Argus X-12 Kit was used to type for X-linked STR (Qiagen GmbH, Hilden, Germany).

    Article Title: Population Genetic Analysis of Haplotypes Based on 17 Short Tandem Repeat Loci on Y Chromosome in Population Sample from Eastern Croatia
    Article Snippet: .. Genomic DNA (1 ng) was amplified using the AmpFISTR Yfiler PCR amplification kit (Applied Biosystems), which coamplifies 17 Y-STRs: DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and GATAH4. .. These amplification reactions were performed using a GeneAmp PCR System 9700 (Applied Biosystems).

    Article Title: Genetic structure in the paternal lineages of South East Spain revealed by the analysis of 17 Y-STRs
    Article Snippet: .. Y-STR genotyping One hundred forty-six samples were amplified using the AmpFl STR® Yfiler® PCR Amplification kit (Applied Biosystems, Foster City, CA), per the manufacturer. .. Alleles were separated and detected on an Applied Biosystems ABI 310 genetic analyzer.

    Article Title: Evaluation of Modified Yfiler(TM) Amplification Strategy for Compromised Samples
    Article Snippet: .. Here, we present experiments conducted in an effort to further characterize the data produced with the AmpFℓSTR® Yfiler™ PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA), using the modified amplification protocol and further explore the potential of Y-STR recovery from a broad range of AFDIL casework material. .. Standard and modified amplifications were conducted using the AmpFℓSTR® Yfiler™ PCR Amplification Kit.

    Article Title: Genetic structure and forensic characteristics of Tibeto-Burman-speaking Ü-Tsang and Kham Tibetan Highlanders revealed by 27 Y-chromosomal STRs
    Article Snippet: .. DNA amplification and genotyping Co-amplification of 27 Y-STRs (DYS576, DYS19, YGATAH4, DYS448, DYS391, DYS456, DYS390, DYS438, DYS392, DYS627, DYS460, DYS458, DYS449, DYS393, DYS518, DYS570, DYS437, DYS385, DYS389I, DYS635, DYS389II, DYS439, DYS481, DYF387S1 and DYS533) was performed using the Yfiler Plus PCR Amplification Kit (Thermo Fisher Scientific) on the ProFlex 3 × 32 PCR System (Thermo Fisher Scientific) according to the manufacturer’s instructions. .. Amplified DNA products were separated and detected using capillary electrophoresis on a 3500XL Genetic Analyzer (Thermo Fisher Scientific) with the LIZ-600 size standard and ABI Hi-DiTM deionized formamide.

    Article Title: Improving global and regional resolution of male lineage differentiation by simple single-copy Y-chromosomal short tandem repeat polymorphisms
    Article Snippet: .. Therefore, more Y-STRs than included in the three commonly-used sets (the 9 Y-STRs comprising the so-called Minimal Haplotype, the 12 Y-STRs included in the PowerPlex Y® System [Promega], or the 17 Y-STRs from the AmpF l STR® Yfiler® PCR Amplification Kit [Applied Biosystems]) are needed for improving the resolution of male lineage differentiation in particular populations, and also for differentiating male relatives in any population. ..

    Article Title: Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR(R) Yfiler(R) PCR amplification kit
    Article Snippet: .. The Y-STRs included in the AmpFl STR® Yfiler® PCR amplification kit (Applied Biosystems, Inc.): DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS348, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4 were genotyped according to the instructions provided by the manufacturer and using a gold-plated silver block GeneAmp®PCR System 9700 (Applied Biosystems, Inc.). .. All PCRs, except for the Berlin samples, were carried out at the Department of Forensic Molecular Biology, Erasmus MC Rotterdam (The Netherlands), and after quality control, PCR products were shipped on dry ice to Applied Biosystems at Foster City (USA), where fragment length analyses was performed using the 3130xl genetic analyzer according to the guidelines in the AmpFl STR® Yfiler® PCR amplification kit user manual.

    Article Title: Molecular genetic identification of skeletal remains from the Second World War Konfin I mass grave in Slovenia
    Article Snippet: .. In persons from the elimination database, in addition to autosomal DNA typing using the AmpFl STR Identifiler™ PCR Amplification Kit (Applied Biosystems), typing of mtDNA was performed, and for males, also typing of Y-STRs using the AmpFl STR Yfiler PCR Amplification Kit (Applied Biosystems). .. MtDNA sequencing The two hypervariable regions HVI and HVII of the mtDNA were amplified by PCR in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

    Article Title: Radiosensitization of head and neck squamous cell carcinoma by a SMAC-mimetic compound, SM-164, requires activation of caspases
    Article Snippet: .. The cell line authentication is as follows: All four HNSCC cell lines were from Dr. T. Carey at the University of Michigan ( ) and have been tested and authenticated by genetic profiling with various microsatellite loci, using Profiler Plus PCR Amplification Kit (Applied Biosystems, Foster City, CA) ( ). ..

    Article Title: A Novel Method of Cell Line Establishment Utilizing Fluorescence-Activated Cell Sorting Resulting in Six New Head and Neck Squamous Cell Carcinoma Lines
    Article Snippet: .. DNA samples were diluted to 0.10 ng/L and were analyzed at the University of Michigan DNA Sequencing Core using the Profiler Plus PCR Amplification Kit (Invitrogen, Carlsbad, California, USA) in accord with the manufacturer’s protocol. ..

    Article Title: Improving global and regional resolution of male lineage differentiation by simple single-copy Y-chromosomal short tandem repeat polymorphisms
    Article Snippet: .. We considered five marker sets: i) all 67 Y-STRs analysed, ii) 49 rarely-studied ssY-STRs, and for comparative reasons the three commonly-used Y-STR sets iii) 17 Y-STRs included in the AmpF l STR® Yfiler® PCR Amplification Kit (Applied Biosystems) (Yfiler), iv) 12 Y-STRs included in the PowerPlex Y® System (Promega) (PPY), and v) 9 Y-STRs comprising the so called Minimal Haplotype (MH). ..

    Isolation:

    Article Title: Population Genetic Analysis of Haplotypes Based on 17 Short Tandem Repeat Loci on Y Chromosome in Population Sample from Eastern Croatia
    Article Snippet: After isolation, the genomic DNA content of each sample was determined by quantitative real-time polymerase chain reaction (PCR) using the Quantifiler™ Human Male DNA Quantification kit (Applied Biosystems, Foster City, CA, USA), which includes an internal positive control to test for the presence of PCR inhibitors in the DNA extracts. .. Genomic DNA (1 ng) was amplified using the AmpFISTR Yfiler PCR amplification kit (Applied Biosystems), which coamplifies 17 Y-STRs: DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and GATAH4.

    Incubation:

    Article Title: Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR(R) Yfiler(R) PCR amplification kit
    Article Snippet: Subsequently, 9-µl reaction buffer plus 1 µl of enzyme mix were added to the cooled sample and incubated at 30°C for 16–18 h, then heat inactivated at 65°C for 10 min. .. The Y-STRs included in the AmpFl STR® Yfiler® PCR amplification kit (Applied Biosystems, Inc.): DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS348, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4 were genotyped according to the instructions provided by the manufacturer and using a gold-plated silver block GeneAmp®PCR System 9700 (Applied Biosystems, Inc.).

    Spectrophotometry:

    Article Title: The genetic evidence for human origin of Jivaroan shrunken heads in collections from the Polish museums
    Article Snippet: The concentration of DNA was determined by spectrophotometry at 260 nm using a GeneQuant RNA/DNA Calculator (Pharmacia Biotech, Cambridge, UK). .. Five commercially available reagent kits for typing of genomic short tandem repeat (STR) markers were used to study nuclear DNA (nDNA): AmpFlSTR Identifiler PCR Amplification Kit, GlobalFiler Amplification Kit, AmpFlSTR YFiler PCR Amplification Kit, and AmpFlSTR YFiler Plus PCR Amplification Kit (all from Applied Biosystems, Foster City, USA).

    Purification:

    Article Title: Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR(R) Yfiler(R) PCR amplification kit
    Article Snippet: Afterwards, the whole-genome-amplified DNA was purified using Invisorb® 96 Filter Microplates (Invitek GmbH, Berlin, Germany). .. The Y-STRs included in the AmpFl STR® Yfiler® PCR amplification kit (Applied Biosystems, Inc.): DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS348, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4 were genotyped according to the instructions provided by the manufacturer and using a gold-plated silver block GeneAmp®PCR System 9700 (Applied Biosystems, Inc.).

    Produced:

    Article Title: Evaluation of Modified Yfiler(TM) Amplification Strategy for Compromised Samples
    Article Snippet: .. Here, we present experiments conducted in an effort to further characterize the data produced with the AmpFℓSTR® Yfiler™ PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA), using the modified amplification protocol and further explore the potential of Y-STR recovery from a broad range of AFDIL casework material. .. Standard and modified amplifications were conducted using the AmpFℓSTR® Yfiler™ PCR Amplification Kit.

    Concentration Assay:

    Article Title: The genetic evidence for human origin of Jivaroan shrunken heads in collections from the Polish museums
    Article Snippet: The concentration of DNA was determined by spectrophotometry at 260 nm using a GeneQuant RNA/DNA Calculator (Pharmacia Biotech, Cambridge, UK). .. Five commercially available reagent kits for typing of genomic short tandem repeat (STR) markers were used to study nuclear DNA (nDNA): AmpFlSTR Identifiler PCR Amplification Kit, GlobalFiler Amplification Kit, AmpFlSTR YFiler PCR Amplification Kit, and AmpFlSTR YFiler Plus PCR Amplification Kit (all from Applied Biosystems, Foster City, USA).

    Article Title: Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR(R) Yfiler(R) PCR amplification kit
    Article Snippet: One or 5 µl (depending on DNA concentration) genomic DNA were added to 9 µl of sample buffer and denatured at 95°C for 3 min, then cooled on ice. .. The Y-STRs included in the AmpFl STR® Yfiler® PCR amplification kit (Applied Biosystems, Inc.): DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS348, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4 were genotyped according to the instructions provided by the manufacturer and using a gold-plated silver block GeneAmp®PCR System 9700 (Applied Biosystems, Inc.).

    Generated:

    Article Title: Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR(R) Yfiler(R) PCR amplification kit
    Article Snippet: The Y-STRs included in the AmpFl STR® Yfiler® PCR amplification kit (Applied Biosystems, Inc.): DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS348, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4 were genotyped according to the instructions provided by the manufacturer and using a gold-plated silver block GeneAmp®PCR System 9700 (Applied Biosystems, Inc.). .. Yfiler profiles were generated using Genemapper ID v3.2 software (Applied Biosystems Inc.), and generated profiles were manually inspected by experienced technicians in Rotterdam for quality control.

    Cell Culture:

    Article Title: Radiosensitization of head and neck squamous cell carcinoma by a SMAC-mimetic compound, SM-164, requires activation of caspases
    Article Snippet: Paragraph title: Cell Culture ... The cell line authentication is as follows: All four HNSCC cell lines were from Dr. T. Carey at the University of Michigan ( ) and have been tested and authenticated by genetic profiling with various microsatellite loci, using Profiler Plus PCR Amplification Kit (Applied Biosystems, Foster City, CA) ( ).

    Touchdown PCR:

    Article Title: Improving global and regional resolution of male lineage differentiation by simple single-copy Y-chromosomal short tandem repeat polymorphisms
    Article Snippet: Initial denaturation was at 95°C for 15 min, followed by 20 cycles of touchdown PCR: 94°C for 30 s, 70°C for 45 s, 72°C for 1 min, with a 1°C decrease in annealing temperature every cycle, and then 15 cycles of standard PCR (94°C for 30 s, 50°C for 45 s, 72°C for 1 min), and finishing with extension at 60°C for 45 min and storage at 4°C. .. In addition, 18 previously used Y-STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y-GATA-H4, and DYS388) of which all except DYS388 were genotyped using the AmpF l STR® Yfiler® PCR amplification kit(Applied Biosystems) according to the instructions provided by the manufacturer.

    Marker:

    Article Title: Improving global and regional resolution of male lineage differentiation by simple single-copy Y-chromosomal short tandem repeat polymorphisms
    Article Snippet: PCR fragment lengths were analyzed by mixing 1 μl of PCR product with 9.7 μl Hi–Di Formamide (Applied Biosystems) and 0.3 μl CXR 60–400 bases size marker (Promega, Madison, WI) and running on 36 cm×50 μm capillaries containing POP-4 polymer (Applied Biosystems) on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). .. In addition, 18 previously used Y-STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y-GATA-H4, and DYS388) of which all except DYS388 were genotyped using the AmpF l STR® Yfiler® PCR amplification kit(Applied Biosystems) according to the instructions provided by the manufacturer.

    Article Title: Improving global and regional resolution of male lineage differentiation by simple single-copy Y-chromosomal short tandem repeat polymorphisms
    Article Snippet: .. We considered five marker sets: i) all 67 Y-STRs analysed, ii) 49 rarely-studied ssY-STRs, and for comparative reasons the three commonly-used Y-STR sets iii) 17 Y-STRs included in the AmpF l STR® Yfiler® PCR Amplification Kit (Applied Biosystems) (Yfiler), iv) 12 Y-STRs included in the PowerPlex Y® System (Promega) (PPY), and v) 9 Y-STRs comprising the so called Minimal Haplotype (MH). ..

    Real-time Polymerase Chain Reaction:

    Article Title: Population Genetic Analysis of Haplotypes Based on 17 Short Tandem Repeat Loci on Y Chromosome in Population Sample from Eastern Croatia
    Article Snippet: Quantitative real-time PCR was performed on a 7500 Real-Time PCR System (Applied Biosystems). .. Genomic DNA (1 ng) was amplified using the AmpFISTR Yfiler PCR amplification kit (Applied Biosystems), which coamplifies 17 Y-STRs: DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and GATAH4.

    Software:

    Article Title: Population Genetic Analysis of Haplotypes Based on 17 Short Tandem Repeat Loci on Y Chromosome in Population Sample from Eastern Croatia
    Article Snippet: Genomic DNA (1 ng) was amplified using the AmpFISTR Yfiler PCR amplification kit (Applied Biosystems), which coamplifies 17 Y-STRs: DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and GATAH4. .. Analysis of the data was performed using Genemapper® software (version 3.2, Applied Biosystems).

    Article Title: Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR(R) Yfiler(R) PCR amplification kit
    Article Snippet: The Y-STRs included in the AmpFl STR® Yfiler® PCR amplification kit (Applied Biosystems, Inc.): DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS348, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4 were genotyped according to the instructions provided by the manufacturer and using a gold-plated silver block GeneAmp®PCR System 9700 (Applied Biosystems, Inc.). .. Yfiler profiles were generated using Genemapper ID v3.2 software (Applied Biosystems Inc.), and generated profiles were manually inspected by experienced technicians in Rotterdam for quality control.

    Article Title: Molecular genetic identification of skeletal remains from the Second World War Konfin I mass grave in Slovenia
    Article Snippet: Genetic profiles were determined using Data Collection v 3.0 and GeneMapper ID v 3.2 (Applied Biosystems) computer software. .. In persons from the elimination database, in addition to autosomal DNA typing using the AmpFl STR Identifiler™ PCR Amplification Kit (Applied Biosystems), typing of mtDNA was performed, and for males, also typing of Y-STRs using the AmpFl STR Yfiler PCR Amplification Kit (Applied Biosystems).

    Article Title: Improving global and regional resolution of male lineage differentiation by simple single-copy Y-chromosomal short tandem repeat polymorphisms
    Article Snippet: Allele sizes were measured using GeneMapper v3.7 software (Applied Biosystems). .. In addition, 18 previously used Y-STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y-GATA-H4, and DYS388) of which all except DYS388 were genotyped using the AmpF l STR® Yfiler® PCR amplification kit(Applied Biosystems) according to the instructions provided by the manufacturer.

    Modification:

    Article Title: Evaluation of Modified Yfiler(TM) Amplification Strategy for Compromised Samples
    Article Snippet: .. Here, we present experiments conducted in an effort to further characterize the data produced with the AmpFℓSTR® Yfiler™ PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA), using the modified amplification protocol and further explore the potential of Y-STR recovery from a broad range of AFDIL casework material. .. Standard and modified amplifications were conducted using the AmpFℓSTR® Yfiler™ PCR Amplification Kit.

    Blocking Assay:

    Article Title: Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR(R) Yfiler(R) PCR amplification kit
    Article Snippet: .. The Y-STRs included in the AmpFl STR® Yfiler® PCR amplification kit (Applied Biosystems, Inc.): DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS348, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4 were genotyped according to the instructions provided by the manufacturer and using a gold-plated silver block GeneAmp®PCR System 9700 (Applied Biosystems, Inc.). .. All PCRs, except for the Berlin samples, were carried out at the Department of Forensic Molecular Biology, Erasmus MC Rotterdam (The Netherlands), and after quality control, PCR products were shipped on dry ice to Applied Biosystems at Foster City (USA), where fragment length analyses was performed using the 3130xl genetic analyzer according to the guidelines in the AmpFl STR® Yfiler® PCR amplification kit user manual.

    DNA Sequencing:

    Article Title: A Novel Method of Cell Line Establishment Utilizing Fluorescence-Activated Cell Sorting Resulting in Six New Head and Neck Squamous Cell Carcinoma Lines
    Article Snippet: .. DNA samples were diluted to 0.10 ng/L and were analyzed at the University of Michigan DNA Sequencing Core using the Profiler Plus PCR Amplification Kit (Invitrogen, Carlsbad, California, USA) in accord with the manufacturer’s protocol. ..

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    Thermo Fisher pcr amplification
    <t>PCR</t> Amplification of 3.3 to 4.2 kb of rDNA from agriculturally important nematodes. M: <t>DNA</t> markers; 1: Heterodera orientalis 104F80; 2: Xiphinema sp. 104F83; 3: Hoplolaimus sp. 104G35; 4: Helicotylenchus sp. 104G36; 5: Meloidogyne incognita Me47; 6: Pratylenchus scribneri Pr1 and 7: Negative control.
    Pcr Amplification, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7702 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PCR Amplification of 3.3 to 4.2 kb of rDNA from agriculturally important nematodes. M: DNA markers; 1: Heterodera orientalis 104F80; 2: Xiphinema sp. 104F83; 3: Hoplolaimus sp. 104G35; 4: Helicotylenchus sp. 104G36; 5: Meloidogyne incognita Me47; 6: Pratylenchus scribneri Pr1 and 7: Negative control.

    Journal: Journal of Nematology

    Article Title: PCR amplification of a long rDNA segment with one primer pair in agriculturally important nematodes

    doi: 10.21307/jofnem-2019-026

    Figure Lengend Snippet: PCR Amplification of 3.3 to 4.2 kb of rDNA from agriculturally important nematodes. M: DNA markers; 1: Heterodera orientalis 104F80; 2: Xiphinema sp. 104F83; 3: Hoplolaimus sp. 104G35; 4: Helicotylenchus sp. 104G36; 5: Meloidogyne incognita Me47; 6: Pratylenchus scribneri Pr1 and 7: Negative control.

    Article Snippet: PCR amplification and DNA Sequencing Each PCR reaction was prepared with 2 µl of DNA extract and 23 µl of the PCR master mix [H2 O: 16.375 µl; 10x DreamTaqTM (Thermo Fisher Scientific, Waltham, MA, USA) buffer: 2.5 µl; dNTP mix, 2.0 mM each: 2.5 µl; 10 µM forward primer: 0.75 µl; 10 µM reverse primer: 0.75 µl; 0.625U DreamTaqTM Hot Start DNA Polymerase 5 U/µl: 0.125 µl, assembled per manufacturer’s manual] containing the primer pairs 18S-CL-F3 and either D3B or 28S-CL-R ( and ).

    Techniques: Polymerase Chain Reaction, Amplification, Negative Control

    Effect of podocin2.5-CCR2 expression on kidney fibronectin and type-1 collagen mRNA expression in diabetic mice Quantitative RT-PCR was performed on whole mouse kidney total RNA after 9 weeks following diabetes. Fibronectin ( A ), and type-1 collagen ( B ) mRNA expression were normalized with GAPDH mRNA. Open bar, control groups; black-filled bar, diabetic groups. Results are means ± SEM. * p

    Journal: Kidney international

    Article Title: Podocyte-specific chemokine (C-C motif) receptor 2 overexpression mediates diabetic renal injury in mice

    doi: 10.1016/j.kint.2016.09.042

    Figure Lengend Snippet: Effect of podocin2.5-CCR2 expression on kidney fibronectin and type-1 collagen mRNA expression in diabetic mice Quantitative RT-PCR was performed on whole mouse kidney total RNA after 9 weeks following diabetes. Fibronectin ( A ), and type-1 collagen ( B ) mRNA expression were normalized with GAPDH mRNA. Open bar, control groups; black-filled bar, diabetic groups. Results are means ± SEM. * p

    Article Snippet: The mouse Ccr2 full-length cDNA was cloned by PCR amplification using kidney RNA template provided by Thermo Scientific (Waltham, MA).

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR

    Effect of podocin2.5-CCR2 expression on kidney inflammatory cytokines in diabetic mice Quantitative RT-PCR was performed on whole mouse kidney total RNA after 9 weeks following diabetes. TNF-α ( A ), and NOS2 ( B ) mRNA expression was normalized with GAPDH mRNA. MSD multi-spot assay system was performed to measure TNF-α (C), and IL-2 (D) protein expression in kidney tissues. Open bar, control groups; black-filled bar, diabetic groups. Results are means ± SEM. * p

    Journal: Kidney international

    Article Title: Podocyte-specific chemokine (C-C motif) receptor 2 overexpression mediates diabetic renal injury in mice

    doi: 10.1016/j.kint.2016.09.042

    Figure Lengend Snippet: Effect of podocin2.5-CCR2 expression on kidney inflammatory cytokines in diabetic mice Quantitative RT-PCR was performed on whole mouse kidney total RNA after 9 weeks following diabetes. TNF-α ( A ), and NOS2 ( B ) mRNA expression was normalized with GAPDH mRNA. MSD multi-spot assay system was performed to measure TNF-α (C), and IL-2 (D) protein expression in kidney tissues. Open bar, control groups; black-filled bar, diabetic groups. Results are means ± SEM. * p

    Article Snippet: The mouse Ccr2 full-length cDNA was cloned by PCR amplification using kidney RNA template provided by Thermo Scientific (Waltham, MA).

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR, Spot Test