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    Structured Review

    Thermo Fisher superscript kit
    Superscript Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript kit/product/Thermo Fisher
    Average 99 stars, based on 84 article reviews
    Price from $9.99 to $1999.99
    superscript kit - by Bioz Stars, 2019-12
    99/100 stars

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    Related Articles

    Countercurrent Chromatography:

    Article Title: COT/MAP3K8 drives resistance to RAF inhibition through MAP kinase pathway reactivation
    Article Snippet: Total mRNA was used for subsequent reverse transcription using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen) for cell lines and unpaired tumor samples, and the SuperScript VILO cDNA synthesis kit (Invitrogen) for paired frozen tumor samples. .. 5 µl of the RT reaction was used for quantitative PCR using SYBR Green PCR Master Mix and gene-specific primers, in triplicate, using an ABI 7300 Real Time PCR System.

    Synthesized:

    Article Title: Induction of Lipocalin2 in a Rat Model of Lung Irradiation
    Article Snippet: RNA content was measured by a spectrophotometer at 260/280 nm. .. In the next step, cDNA was synthesized by reverse transcription of isolated RNA utilizing the commercial superscript kit (Invitrogen, Groningen, The Netherlands), according to the manufacturer’s instructions. .. The targeted genes were amplified in the Step one Plus real-time PCR cycler (Applied Biosystems, Darmstadt, Germany) at 95–60 °C for 40 thermal cycles.

    Isolation:

    Article Title: Induction of Lipocalin2 in a Rat Model of Lung Irradiation
    Article Snippet: RNA content was measured by a spectrophotometer at 260/280 nm. .. In the next step, cDNA was synthesized by reverse transcription of isolated RNA utilizing the commercial superscript kit (Invitrogen, Groningen, The Netherlands), according to the manufacturer’s instructions. .. The targeted genes were amplified in the Step one Plus real-time PCR cycler (Applied Biosystems, Darmstadt, Germany) at 95–60 °C for 40 thermal cycles.

    Spectrophotometry:

    Article Title: Induction of Lipocalin2 in a Rat Model of Lung Irradiation
    Article Snippet: RNA content was measured by a spectrophotometer at 260/280 nm. .. In the next step, cDNA was synthesized by reverse transcription of isolated RNA utilizing the commercial superscript kit (Invitrogen, Groningen, The Netherlands), according to the manufacturer’s instructions.

    SYBR Green Assay:

    Article Title: Induction of Lipocalin2 in a Rat Model of Lung Irradiation
    Article Snippet: In the next step, cDNA was synthesized by reverse transcription of isolated RNA utilizing the commercial superscript kit (Invitrogen, Groningen, The Netherlands), according to the manufacturer’s instructions. .. The targeted genes were amplified in the Step one Plus real-time PCR cycler (Applied Biosystems, Darmstadt, Germany) at 95–60 °C for 40 thermal cycles.

    Expressing:

    Article Title: Induction of Lipocalin2 in a Rat Model of Lung Irradiation
    Article Snippet: In the next step, cDNA was synthesized by reverse transcription of isolated RNA utilizing the commercial superscript kit (Invitrogen, Groningen, The Netherlands), according to the manufacturer’s instructions. .. SYBR Green UDG reaction master mix (Invitrogen Groningen, The Netherlands) served for relative quantification of cDNA.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: COT/MAP3K8 drives resistance to RAF inhibition through MAP kinase pathway reactivation
    Article Snippet: Paragraph title: Quantitative RT/PCR ... Total mRNA was used for subsequent reverse transcription using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen) for cell lines and unpaired tumor samples, and the SuperScript VILO cDNA synthesis kit (Invitrogen) for paired frozen tumor samples.

    Article Title: Induction of Lipocalin2 in a Rat Model of Lung Irradiation
    Article Snippet: Paragraph title: 4.4. RNA Isolation and RT-PCR ... In the next step, cDNA was synthesized by reverse transcription of isolated RNA utilizing the commercial superscript kit (Invitrogen, Groningen, The Netherlands), according to the manufacturer’s instructions.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: COT/MAP3K8 drives resistance to RAF inhibition through MAP kinase pathway reactivation
    Article Snippet: Total mRNA was used for subsequent reverse transcription using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen) for cell lines and unpaired tumor samples, and the SuperScript VILO cDNA synthesis kit (Invitrogen) for paired frozen tumor samples. .. 5 µl of the RT reaction was used for quantitative PCR using SYBR Green PCR Master Mix and gene-specific primers, in triplicate, using an ABI 7300 Real Time PCR System.

    Cellular Antioxidant Activity Assay:

    Article Title: COT/MAP3K8 drives resistance to RAF inhibition through MAP kinase pathway reactivation
    Article Snippet: Total mRNA was used for subsequent reverse transcription using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen) for cell lines and unpaired tumor samples, and the SuperScript VILO cDNA synthesis kit (Invitrogen) for paired frozen tumor samples. .. 5 µl of the RT reaction was used for quantitative PCR using SYBR Green PCR Master Mix and gene-specific primers, in triplicate, using an ABI 7300 Real Time PCR System.

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    Thermo Fisher superscript iii kit
    The FEN1 inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. <t>qPCR</t> analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of <t>three</t> independent experiments. Asterisks indicate statistically significant differences; * P
    Superscript Iii Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 328 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii kit/product/Thermo Fisher
    Average 99 stars, based on 328 article reviews
    Price from $9.99 to $1999.99
    superscript iii kit - by Bioz Stars, 2019-12
    99/100 stars
      Buy from Supplier

    80
    Thermo Fisher superscript iii first strand synthesis system
    BRCA2 deficiency causes <t>DNA</t> under replication that results in abnormal mitoses. a – c BRCA2 ∆Ex3-4/– cells were released for 22 h from serum starvation to increase mitotic cells, incubated with EdU for 1 h, and then analyzed for mitotic DNA synthesis. Early mitotic cells defined as being in prophase, prometaphase, or metaphase were analyzed for EdU foci that co-localize with FANCD2 foci pairs. a Representative images of mitotic DNA synthesis. Scale bars 10 µm. b Percent early mitotic cells containing EdU foci, analyzed by an unpaired two-tailed t -test. n = 3. c FANCD2 foci pairs with or without EdU foci co-localization. Graphs represent the pooled results of <t>three</t> independent experiments, each analyzed by a two-tailed Mann–Whitney test. Median FANCD2 foci pair number, red bars . d FANCD2 foci pair analysis in early mitotic cells. Cells were untreated, or treated with the CDK1 inhibitor RO-3306 (10 µM, 24 h) to delay mitotic entry, and released for 1 h before analysis of FANCD2 foci pairs in early mitotic cells. Analysis is by an unpaired two-tailed t -test. n = 4. e – h Anaphase cells were analyzed for DAPI bridges ( e , g ) and lagging chromosomes ( f , h ). RO-3306 treatment (10 µM, 24 h with release for 1 h) was applied where indicated. Representative deconvolved images are shown on the left of e and f . n = 3. Statistical analysis was by an unpaired two-tailed t -test. FD2, FANCD2. Scale bars 10 µm. i , j . 53BP1 nuclear body analysis with RO-3306 (10 µM, i ) or nocodazole (100 ng ml −1 , j ) treatment and released as in the schematic on the left . The fraction of EdU+ cells in G1 at the time of harvest that contains ≥3 53BP1 nuclear bodies (NBs) is shown in the graph on the right . Analysis is by an unpaired two-tailed t -test. n ≥ 3. Error bars s.d. ns, not significant; * p
    Superscript Iii First Strand Synthesis System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii first strand synthesis system/product/Thermo Fisher
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    superscript iii first strand synthesis system - by Bioz Stars, 2019-12
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    Image Search Results


    The FEN1 inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: The FEN1 inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Article Snippet: Total RNA was treated with amplification grade DNase I (Thermo Fisher Scientific) and reverse transcribed using an oligo (dT) primer and the SuperScript III kit (Thermo Fisher Scientific). qPCR analysis of resulting cDNA was performed using SYBR green ROX (Toyobo) with MX3000 (Stratagene) as described previously [ ].

    Techniques: Inhibition, Real-time Polymerase Chain Reaction

    Requirement of nuclease activity and the C-terminus of FEN1 for cccDNA production. (A) Schematic presentation of FEN1 protein. D181A: nuclease-deficient mutant. ΔC: deletion mutant unable to bind WRN protein. (B) FEN assay. Flap endonuclease activity of immunoprecipitated FEN1 protein (wt, D181A, or ΔC) was determined as in Fig 1A . (C) pResQ lentiviral vectors carrying FEN1 shRNA and the FEN1 transgene (wt, D181A, or ΔC, see also S8 Fig ) were transduced into Hep38.7-Tet cells. After puromycin selection, NC-DNA was analyzed by Southern blotting and qPCR, respectively. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; **** P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: Requirement of nuclease activity and the C-terminus of FEN1 for cccDNA production. (A) Schematic presentation of FEN1 protein. D181A: nuclease-deficient mutant. ΔC: deletion mutant unable to bind WRN protein. (B) FEN assay. Flap endonuclease activity of immunoprecipitated FEN1 protein (wt, D181A, or ΔC) was determined as in Fig 1A . (C) pResQ lentiviral vectors carrying FEN1 shRNA and the FEN1 transgene (wt, D181A, or ΔC, see also S8 Fig ) were transduced into Hep38.7-Tet cells. After puromycin selection, NC-DNA was analyzed by Southern blotting and qPCR, respectively. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; **** P

    Article Snippet: Total RNA was treated with amplification grade DNase I (Thermo Fisher Scientific) and reverse transcribed using an oligo (dT) primer and the SuperScript III kit (Thermo Fisher Scientific). qPCR analysis of resulting cDNA was performed using SYBR green ROX (Toyobo) with MX3000 (Stratagene) as described previously [ ].

    Techniques: Activity Assay, Mutagenesis, Immunoprecipitation, shRNA, Selection, Southern Blot, Real-time Polymerase Chain Reaction

    Deletion of the C-terminus disrupts the nuclear localization and reduces HBV DNA association of FEN1 protein. (A) Expression vectors of FEN1-GFP, FEN1ΔC-GFP, or mock vector (pcDNA4/TO) were transfected into Hep38.7-Tet cells. The nucleus was visualized by co-transfection of the nuclear localization signal (NLS)-tagged DsRed vector. (B–C) Myc-FEN1 or Myc-FEN1ΔC vector was transfected into Hep38.7-Tet cells. (B) Myc-tagged protein expression (before cross linkage) shown by Western blot. Two blots with different protein loadings are shown. (C) Myc-FEN1-transfected Hep38.7-Tet cells were cross-linked, and the lysates were immunoprecipitated with either control IgG or anti-Myc antibody. The immunoprecipitants were subjected to qPCR analysis using a primer pair to detect the core region of HBV. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: Deletion of the C-terminus disrupts the nuclear localization and reduces HBV DNA association of FEN1 protein. (A) Expression vectors of FEN1-GFP, FEN1ΔC-GFP, or mock vector (pcDNA4/TO) were transfected into Hep38.7-Tet cells. The nucleus was visualized by co-transfection of the nuclear localization signal (NLS)-tagged DsRed vector. (B–C) Myc-FEN1 or Myc-FEN1ΔC vector was transfected into Hep38.7-Tet cells. (B) Myc-tagged protein expression (before cross linkage) shown by Western blot. Two blots with different protein loadings are shown. (C) Myc-FEN1-transfected Hep38.7-Tet cells were cross-linked, and the lysates were immunoprecipitated with either control IgG or anti-Myc antibody. The immunoprecipitants were subjected to qPCR analysis using a primer pair to detect the core region of HBV. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Article Snippet: Total RNA was treated with amplification grade DNase I (Thermo Fisher Scientific) and reverse transcribed using an oligo (dT) primer and the SuperScript III kit (Thermo Fisher Scientific). qPCR analysis of resulting cDNA was performed using SYBR green ROX (Toyobo) with MX3000 (Stratagene) as described previously [ ].

    Techniques: Expressing, Plasmid Preparation, Transfection, Cotransfection, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction

    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Article Snippet: Total RNA was treated with amplification grade DNase I (Thermo Fisher Scientific) and reverse transcribed using an oligo (dT) primer and the SuperScript III kit (Thermo Fisher Scientific). qPCR analysis of resulting cDNA was performed using SYBR green ROX (Toyobo) with MX3000 (Stratagene) as described previously [ ].

    Techniques: In Vitro, Tube Formation Assay, Purification, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing

    FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production. (A–D) Hep38.7-Tet cells were transfected with FEN1 -specific siRNA (siFEN1 #1 or #2) or control (siCtrl), and cultured without tetracycline. Four days after transfection, FEN1 mRNA/protein and HBV DNA were analyzed. (A) FEN1 mRNA quantified by RT-qPCR (normalized by HPRT) (upper panel) and Western blotting of FEN1 protein (lower panel). GAPDH expression is shown as a loading control. FEN1 protein expression gives rise to two bands in our study, which may be due to post-translational modification. (B–C) Levels of HBV DNA in cytoplasmic nucleocapsid (NC) (B) and cccDNA (C). (D) Efficiency of cccDNA formation (cccDNA levels normalized by cytoplasmic NC-DNA) was calculated. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production. (A–D) Hep38.7-Tet cells were transfected with FEN1 -specific siRNA (siFEN1 #1 or #2) or control (siCtrl), and cultured without tetracycline. Four days after transfection, FEN1 mRNA/protein and HBV DNA were analyzed. (A) FEN1 mRNA quantified by RT-qPCR (normalized by HPRT) (upper panel) and Western blotting of FEN1 protein (lower panel). GAPDH expression is shown as a loading control. FEN1 protein expression gives rise to two bands in our study, which may be due to post-translational modification. (B–C) Levels of HBV DNA in cytoplasmic nucleocapsid (NC) (B) and cccDNA (C). (D) Efficiency of cccDNA formation (cccDNA levels normalized by cytoplasmic NC-DNA) was calculated. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Article Snippet: Total RNA was treated with amplification grade DNase I (Thermo Fisher Scientific) and reverse transcribed using an oligo (dT) primer and the SuperScript III kit (Thermo Fisher Scientific). qPCR analysis of resulting cDNA was performed using SYBR green ROX (Toyobo) with MX3000 (Stratagene) as described previously [ ].

    Techniques: CRISPR, Transfection, Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Modification

    Global changes in infected MDM gene expression. MDMs from four healthy human donors were infected at a 2:1 (parasite:MDM) ratio with each of three isolates of L. braziliensis from clades A, B, or C ( Schriefer et al., 2004 ). The clades A, B, and C isolates were drawn from patients with DL, CL, and ML, respectively. After 4 h, total RNA was extracted and processed for hybridization to Affymetrix human transcript microarrays. Fold changes were calculated by comparing fluorescence data representing the abundance of each transcript in infected versus uninfected MDMs from the same donor. Each dot in the figure represents the average fold change in abundance of each transcript in all four donors. Eighty-nine transcripts were significantly increased, and 471 transcripts were significantly decreased after MDM infection with each of the three L. braziliensis isolates (one-way ANOVA for repressed transcripts among MDM infected with clades A, B, or C, p

    Journal: Frontiers in Microbiology

    Article Title: Early Suppression of Macrophage Gene Expression by Leishmania braziliensis

    doi: 10.3389/fmicb.2018.02464

    Figure Lengend Snippet: Global changes in infected MDM gene expression. MDMs from four healthy human donors were infected at a 2:1 (parasite:MDM) ratio with each of three isolates of L. braziliensis from clades A, B, or C ( Schriefer et al., 2004 ). The clades A, B, and C isolates were drawn from patients with DL, CL, and ML, respectively. After 4 h, total RNA was extracted and processed for hybridization to Affymetrix human transcript microarrays. Fold changes were calculated by comparing fluorescence data representing the abundance of each transcript in infected versus uninfected MDMs from the same donor. Each dot in the figure represents the average fold change in abundance of each transcript in all four donors. Eighty-nine transcripts were significantly increased, and 471 transcripts were significantly decreased after MDM infection with each of the three L. braziliensis isolates (one-way ANOVA for repressed transcripts among MDM infected with clades A, B, or C, p

    Article Snippet: Changes in expression observed on microarrays were validated by reverse transcriptase followed by qPCR to document gene expression in RNA samples from replicate MDM samples incubated without or with the representatives of L. braziliensis clades. cDNA was generated using the Superscript III First Strand Synthesis System kit (Invitrogen/ThermoFisher, Waltham, MA, United States) and random hexamers, followed by RNase H treatment according to the manufacturer’s instructions.

    Techniques: Infection, Expressing, Hybridization, Fluorescence

    Gene expression profiles from the microarrays described and illustrated in Figure 2 were collated according to the number of significantly altered transcripts in MDM infected with isolates from each of the three representatives of L. braziliensis clades (A, B, or C). (A) Venn diagram of the distribution of transcripts with changes in expression that reached statistical significance upon infection of MDMs with each L. braziliensis parasite. Sectors indicate the numbers of transcripts that were uniquely changed due to infection with one parasite clade, or transcripts that were changed by infection with more than one parasite clade (evaluation of gene expression employed ANOVA for detecting transcripts significantly affected by infections, and paired Student’s t -test for comparing the expression elicited by clades of parasites in infected MDM). (B) The magnitude of change in expression of 471 genes in MDM infected with each of the three L. braziliensis isolates is illustrated. Values represent the fold changes in expression of the 471 genes for which transcript abundance was significantly decreased by infection with any of the three parasite isolates tested. L. braziliensis isolates belong to clade A (DL; blue), clade B (CL; green), or clade C (ML; red). Each position on the x-axis corresponds to a single gene, plotted against its fold change in expression on the y-axis (Friedman’s test p

    Journal: Frontiers in Microbiology

    Article Title: Early Suppression of Macrophage Gene Expression by Leishmania braziliensis

    doi: 10.3389/fmicb.2018.02464

    Figure Lengend Snippet: Gene expression profiles from the microarrays described and illustrated in Figure 2 were collated according to the number of significantly altered transcripts in MDM infected with isolates from each of the three representatives of L. braziliensis clades (A, B, or C). (A) Venn diagram of the distribution of transcripts with changes in expression that reached statistical significance upon infection of MDMs with each L. braziliensis parasite. Sectors indicate the numbers of transcripts that were uniquely changed due to infection with one parasite clade, or transcripts that were changed by infection with more than one parasite clade (evaluation of gene expression employed ANOVA for detecting transcripts significantly affected by infections, and paired Student’s t -test for comparing the expression elicited by clades of parasites in infected MDM). (B) The magnitude of change in expression of 471 genes in MDM infected with each of the three L. braziliensis isolates is illustrated. Values represent the fold changes in expression of the 471 genes for which transcript abundance was significantly decreased by infection with any of the three parasite isolates tested. L. braziliensis isolates belong to clade A (DL; blue), clade B (CL; green), or clade C (ML; red). Each position on the x-axis corresponds to a single gene, plotted against its fold change in expression on the y-axis (Friedman’s test p

    Article Snippet: Changes in expression observed on microarrays were validated by reverse transcriptase followed by qPCR to document gene expression in RNA samples from replicate MDM samples incubated without or with the representatives of L. braziliensis clades. cDNA was generated using the Superscript III First Strand Synthesis System kit (Invitrogen/ThermoFisher, Waltham, MA, United States) and random hexamers, followed by RNase H treatment according to the manufacturer’s instructions.

    Techniques: Expressing, Infection

    Changes in LRRK2, TLR8, HSPA1A, and MT1M expression were documented in independent assays of MDMs from eight additional human donors, distinct from those of experiments depicted in Figures 1 – 4 . MDMs were infected with three representative L. braziliensis isolates of each clade (A, B, or C) used in microarray experiments. Data derived from total RNA extracted after 4 h of MDM infection at a MOI of two parasites per macrophage (2:1). The relative abundance of transcripts was assessed by RT-qPCR. p -Values correspond to pair-wise comparisons by one tailed paired Wilcoxon test.

    Journal: Frontiers in Microbiology

    Article Title: Early Suppression of Macrophage Gene Expression by Leishmania braziliensis

    doi: 10.3389/fmicb.2018.02464

    Figure Lengend Snippet: Changes in LRRK2, TLR8, HSPA1A, and MT1M expression were documented in independent assays of MDMs from eight additional human donors, distinct from those of experiments depicted in Figures 1 – 4 . MDMs were infected with three representative L. braziliensis isolates of each clade (A, B, or C) used in microarray experiments. Data derived from total RNA extracted after 4 h of MDM infection at a MOI of two parasites per macrophage (2:1). The relative abundance of transcripts was assessed by RT-qPCR. p -Values correspond to pair-wise comparisons by one tailed paired Wilcoxon test.

    Article Snippet: Changes in expression observed on microarrays were validated by reverse transcriptase followed by qPCR to document gene expression in RNA samples from replicate MDM samples incubated without or with the representatives of L. braziliensis clades. cDNA was generated using the Superscript III First Strand Synthesis System kit (Invitrogen/ThermoFisher, Waltham, MA, United States) and random hexamers, followed by RNase H treatment according to the manufacturer’s instructions.

    Techniques: Expressing, Infection, Microarray, Derivative Assay, Quantitative RT-PCR, One-tailed Test

    BRCA2 deficiency causes DNA under replication that results in abnormal mitoses. a – c BRCA2 ∆Ex3-4/– cells were released for 22 h from serum starvation to increase mitotic cells, incubated with EdU for 1 h, and then analyzed for mitotic DNA synthesis. Early mitotic cells defined as being in prophase, prometaphase, or metaphase were analyzed for EdU foci that co-localize with FANCD2 foci pairs. a Representative images of mitotic DNA synthesis. Scale bars 10 µm. b Percent early mitotic cells containing EdU foci, analyzed by an unpaired two-tailed t -test. n = 3. c FANCD2 foci pairs with or without EdU foci co-localization. Graphs represent the pooled results of three independent experiments, each analyzed by a two-tailed Mann–Whitney test. Median FANCD2 foci pair number, red bars . d FANCD2 foci pair analysis in early mitotic cells. Cells were untreated, or treated with the CDK1 inhibitor RO-3306 (10 µM, 24 h) to delay mitotic entry, and released for 1 h before analysis of FANCD2 foci pairs in early mitotic cells. Analysis is by an unpaired two-tailed t -test. n = 4. e – h Anaphase cells were analyzed for DAPI bridges ( e , g ) and lagging chromosomes ( f , h ). RO-3306 treatment (10 µM, 24 h with release for 1 h) was applied where indicated. Representative deconvolved images are shown on the left of e and f . n = 3. Statistical analysis was by an unpaired two-tailed t -test. FD2, FANCD2. Scale bars 10 µm. i , j . 53BP1 nuclear body analysis with RO-3306 (10 µM, i ) or nocodazole (100 ng ml −1 , j ) treatment and released as in the schematic on the left . The fraction of EdU+ cells in G1 at the time of harvest that contains ≥3 53BP1 nuclear bodies (NBs) is shown in the graph on the right . Analysis is by an unpaired two-tailed t -test. n ≥ 3. Error bars s.d. ns, not significant; * p

    Journal: Nature Communications

    Article Title: BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination

    doi: 10.1038/s41467-017-00634-0

    Figure Lengend Snippet: BRCA2 deficiency causes DNA under replication that results in abnormal mitoses. a – c BRCA2 ∆Ex3-4/– cells were released for 22 h from serum starvation to increase mitotic cells, incubated with EdU for 1 h, and then analyzed for mitotic DNA synthesis. Early mitotic cells defined as being in prophase, prometaphase, or metaphase were analyzed for EdU foci that co-localize with FANCD2 foci pairs. a Representative images of mitotic DNA synthesis. Scale bars 10 µm. b Percent early mitotic cells containing EdU foci, analyzed by an unpaired two-tailed t -test. n = 3. c FANCD2 foci pairs with or without EdU foci co-localization. Graphs represent the pooled results of three independent experiments, each analyzed by a two-tailed Mann–Whitney test. Median FANCD2 foci pair number, red bars . d FANCD2 foci pair analysis in early mitotic cells. Cells were untreated, or treated with the CDK1 inhibitor RO-3306 (10 µM, 24 h) to delay mitotic entry, and released for 1 h before analysis of FANCD2 foci pairs in early mitotic cells. Analysis is by an unpaired two-tailed t -test. n = 4. e – h Anaphase cells were analyzed for DAPI bridges ( e , g ) and lagging chromosomes ( f , h ). RO-3306 treatment (10 µM, 24 h with release for 1 h) was applied where indicated. Representative deconvolved images are shown on the left of e and f . n = 3. Statistical analysis was by an unpaired two-tailed t -test. FD2, FANCD2. Scale bars 10 µm. i , j . 53BP1 nuclear body analysis with RO-3306 (10 µM, i ) or nocodazole (100 ng ml −1 , j ) treatment and released as in the schematic on the left . The fraction of EdU+ cells in G1 at the time of harvest that contains ≥3 53BP1 nuclear bodies (NBs) is shown in the graph on the right . Analysis is by an unpaired two-tailed t -test. n ≥ 3. Error bars s.d. ns, not significant; * p

    Article Snippet: Complementary DNA was then synthesized from RNA using SuperScript® III First-Strand Synthesis Kit (18080051, Thermo Fisher Scientific) following the manufacturer’s instructions.

    Techniques: Incubation, DNA Synthesis, Two Tailed Test, MANN-WHITNEY

    Fork protection is a minor replication stress suppression pathway. a Analysis of mitotic DNA synthesis in BRCA2 –/– AAVS1 ∆ cells complemented by BRCA2 WT or BRCA2 SE. Cells were released for 22 h from serum starvation to increase mitotic cells, incubated with EdU 1 h, and then analyzed for mitotic DNA synthesis. Early mitotic cells, defined as being in prophase, prometaphase, or metaphase, were analyzed for EdU foci that co-localize with FANCD2 foci pairs. The percent early mitotic cells containing EdU foci ( left , n = 3) or EdU foci distribution in these cells ( right , pooled results of three independent experiments) was quantified. b , c Spontaneous 53BP1 nuclear body analysis in G1 in both BRCA2 ∆Ex3-4/– cells ( b ) and BRCA2 –/– AAVS1 ∆ cells ( c ) complemented by BRCA2 WT or BRCA2 SE. n ≥ 3. d – g HU-induced 53BP1 nuclear body formation analysis, as in Fig. 4d , using complemented BRCA2 ∆Ex3-4/– cells ( d ) or BRCA2 –/ – AAVS1 ∆ cells ( e ), stable MRE11 or PARP1 knockdown BRCA2 ∆Ex3-4/ – cells ( f ), and BRCA2 ∆Ex3-4/ – cells with the indicated PARP1 genotype ( g ). n ≥ 3. Error bars s.d. ns, not significant; * p

    Journal: Nature Communications

    Article Title: BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination

    doi: 10.1038/s41467-017-00634-0

    Figure Lengend Snippet: Fork protection is a minor replication stress suppression pathway. a Analysis of mitotic DNA synthesis in BRCA2 –/– AAVS1 ∆ cells complemented by BRCA2 WT or BRCA2 SE. Cells were released for 22 h from serum starvation to increase mitotic cells, incubated with EdU 1 h, and then analyzed for mitotic DNA synthesis. Early mitotic cells, defined as being in prophase, prometaphase, or metaphase, were analyzed for EdU foci that co-localize with FANCD2 foci pairs. The percent early mitotic cells containing EdU foci ( left , n = 3) or EdU foci distribution in these cells ( right , pooled results of three independent experiments) was quantified. b , c Spontaneous 53BP1 nuclear body analysis in G1 in both BRCA2 ∆Ex3-4/– cells ( b ) and BRCA2 –/– AAVS1 ∆ cells ( c ) complemented by BRCA2 WT or BRCA2 SE. n ≥ 3. d – g HU-induced 53BP1 nuclear body formation analysis, as in Fig. 4d , using complemented BRCA2 ∆Ex3-4/– cells ( d ) or BRCA2 –/ – AAVS1 ∆ cells ( e ), stable MRE11 or PARP1 knockdown BRCA2 ∆Ex3-4/ – cells ( f ), and BRCA2 ∆Ex3-4/ – cells with the indicated PARP1 genotype ( g ). n ≥ 3. Error bars s.d. ns, not significant; * p

    Article Snippet: Complementary DNA was then synthesized from RNA using SuperScript® III First-Strand Synthesis Kit (18080051, Thermo Fisher Scientific) following the manufacturer’s instructions.

    Techniques: DNA Synthesis, Incubation

    HR proficiency is associated with replication stress suppression and cell viability. a Western blot showing RAD51 knockdown in BRCA2 ∆Ex3-4/ – cells stably expressing BRCA2 WT or BRCA2 SE. NT, non targeting siRNA. b HR analysis. Cells expressing RAD51 siRNAs were infected with I-SceI-expressing lentivirus and the percent GFP+ cells was analyzed by an unpaired two-tailed t -test. n ≥ 3. c Cells expressing RAD51 siRNAs were analyzed for fork protection in the presence of HU. Median CldU/IdU tract length ratios are indicated in the graph ( red bars ). Graphs represent the pooled results of > 300 fibers per genotype from at least three independent experiments, analyzed by a two-tailed Mann–Whitney test. d Cells expressing RAD51 siRNAs were released for 22 h from serum starvation to increase mitotic cells, incubated with EdU 1 h, and then analyzed for mitotic DNA synthesis. Early mitotic cells, defined as being in prophase, prometaphase, or metaphase, were analyzed for EdU foci that co-localize with FANCD2 foci pairs. The percent early mitotic cells containing EdU foci were analyzed by an unpaired two-tailed t -test. n = 3. e HU-induced 53BP1 nuclear body formation analysis, as in Fig. 4d , using cells expressing RAD51 siRNAs. Statistical analysis was by an unpaired two-tailed t -test. n ≥ 4. f RAD51 depletion in BRCA2 ∆Ex3-4/ – cells stably expressing BRCA2 WT or BRCA2 SE leads to a reduction in clonogenic survival. g HU-induced 53BP1 nuclear body formation analysis, as in Fig. 4d , using cells transfected with RAD51 siRNAs. Statistical analysis was by an unpaired two-tailed t -test. n = 4. Error bars s.d. ns, not significant; * p

    Journal: Nature Communications

    Article Title: BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination

    doi: 10.1038/s41467-017-00634-0

    Figure Lengend Snippet: HR proficiency is associated with replication stress suppression and cell viability. a Western blot showing RAD51 knockdown in BRCA2 ∆Ex3-4/ – cells stably expressing BRCA2 WT or BRCA2 SE. NT, non targeting siRNA. b HR analysis. Cells expressing RAD51 siRNAs were infected with I-SceI-expressing lentivirus and the percent GFP+ cells was analyzed by an unpaired two-tailed t -test. n ≥ 3. c Cells expressing RAD51 siRNAs were analyzed for fork protection in the presence of HU. Median CldU/IdU tract length ratios are indicated in the graph ( red bars ). Graphs represent the pooled results of > 300 fibers per genotype from at least three independent experiments, analyzed by a two-tailed Mann–Whitney test. d Cells expressing RAD51 siRNAs were released for 22 h from serum starvation to increase mitotic cells, incubated with EdU 1 h, and then analyzed for mitotic DNA synthesis. Early mitotic cells, defined as being in prophase, prometaphase, or metaphase, were analyzed for EdU foci that co-localize with FANCD2 foci pairs. The percent early mitotic cells containing EdU foci were analyzed by an unpaired two-tailed t -test. n = 3. e HU-induced 53BP1 nuclear body formation analysis, as in Fig. 4d , using cells expressing RAD51 siRNAs. Statistical analysis was by an unpaired two-tailed t -test. n ≥ 4. f RAD51 depletion in BRCA2 ∆Ex3-4/ – cells stably expressing BRCA2 WT or BRCA2 SE leads to a reduction in clonogenic survival. g HU-induced 53BP1 nuclear body formation analysis, as in Fig. 4d , using cells transfected with RAD51 siRNAs. Statistical analysis was by an unpaired two-tailed t -test. n = 4. Error bars s.d. ns, not significant; * p

    Article Snippet: Complementary DNA was then synthesized from RNA using SuperScript® III First-Strand Synthesis Kit (18080051, Thermo Fisher Scientific) following the manufacturer’s instructions.

    Techniques: Western Blot, Stable Transfection, Expressing, Infection, Two Tailed Test, MANN-WHITNEY, Incubation, DNA Synthesis, Transfection

    Fork protection plays a minor role in cell viability and DNA repair. a DNA fiber analysis to quantify fork protection. Schematic of the experimental design and representative images are shown in the inset . Median CldU/IdU tract length ratios are indicated in the graph ( red bars ). Graphs here and below represent the pooled results of > 200 fibers per genotype from at least two independent experiments, analyzed by a two-tailed Mann–Whitney test. b Cells stably expressing the indicated shRNAs were analyzed for fork protection in the presence of HU by a two-tailed Mann–Whitney test. Scr, scrambled shRNA. c HR analysis. PARP1 knockdown cells were infected with I-SceI-expressing lentivirus and the percent GFP+ cells was analyzed by an unpaired two-tailed t -test. n = 3. d PARP1 knockdown cells were plated for clonogenic survival. Residual colonies from BRCA2 ∆Ex3-4/− plates were all were confirmed by PCR to have maintained the BRCA2 fl/− genotype (i.e., escaped Cre recombination). e Cisplatin-induced γH2AX. PARP1 knockdown cells were treated with 5 µM cisplatin for 5 h and released for another 24 h before analysis. γH2AX mean nuclear intensities of > 1000 individual cells are shown from one experiment, which is representative of three independent experiments, analyzed by a two-tailed Mann–Whitney test. The dotted red line indicates the median of BRCA2 mutant cells treated with the scrambled shRNA exposed to cisplatin. A.U., arbitrary units. Box and whiskers show the 10th and 90th percentiles. f Western blotting of BRCA2 −/− AAVS1 fl cells stably expressing BRCA2 WT or BRCA2 SE. g DNA fiber analysis to quantify fork protection in the presence of HU. Median CldU/IdU tract length ratios are indicated ( red bars ), analyzed by a two-tailed Mann–Whitney test. h HR analysis, as in c . n > 3. i Clonogenic survival analysis using an unpaired two-tailed t -test. n = 3. j Cisplatin-induced γH2AX analysis, as in e . The dotted red line indicates the median of the BRCA2 −/− AAVS1 ∆ cells exposed to cisplatin. Error bars s.d. ns, not significant; * p

    Journal: Nature Communications

    Article Title: BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination

    doi: 10.1038/s41467-017-00634-0

    Figure Lengend Snippet: Fork protection plays a minor role in cell viability and DNA repair. a DNA fiber analysis to quantify fork protection. Schematic of the experimental design and representative images are shown in the inset . Median CldU/IdU tract length ratios are indicated in the graph ( red bars ). Graphs here and below represent the pooled results of > 200 fibers per genotype from at least two independent experiments, analyzed by a two-tailed Mann–Whitney test. b Cells stably expressing the indicated shRNAs were analyzed for fork protection in the presence of HU by a two-tailed Mann–Whitney test. Scr, scrambled shRNA. c HR analysis. PARP1 knockdown cells were infected with I-SceI-expressing lentivirus and the percent GFP+ cells was analyzed by an unpaired two-tailed t -test. n = 3. d PARP1 knockdown cells were plated for clonogenic survival. Residual colonies from BRCA2 ∆Ex3-4/− plates were all were confirmed by PCR to have maintained the BRCA2 fl/− genotype (i.e., escaped Cre recombination). e Cisplatin-induced γH2AX. PARP1 knockdown cells were treated with 5 µM cisplatin for 5 h and released for another 24 h before analysis. γH2AX mean nuclear intensities of > 1000 individual cells are shown from one experiment, which is representative of three independent experiments, analyzed by a two-tailed Mann–Whitney test. The dotted red line indicates the median of BRCA2 mutant cells treated with the scrambled shRNA exposed to cisplatin. A.U., arbitrary units. Box and whiskers show the 10th and 90th percentiles. f Western blotting of BRCA2 −/− AAVS1 fl cells stably expressing BRCA2 WT or BRCA2 SE. g DNA fiber analysis to quantify fork protection in the presence of HU. Median CldU/IdU tract length ratios are indicated ( red bars ), analyzed by a two-tailed Mann–Whitney test. h HR analysis, as in c . n > 3. i Clonogenic survival analysis using an unpaired two-tailed t -test. n = 3. j Cisplatin-induced γH2AX analysis, as in e . The dotted red line indicates the median of the BRCA2 −/− AAVS1 ∆ cells exposed to cisplatin. Error bars s.d. ns, not significant; * p

    Article Snippet: Complementary DNA was then synthesized from RNA using SuperScript® III First-Strand Synthesis Kit (18080051, Thermo Fisher Scientific) following the manufacturer’s instructions.

    Techniques: Two Tailed Test, MANN-WHITNEY, Stable Transfection, Expressing, shRNA, Infection, Polymerase Chain Reaction, Mutagenesis, Western Blot

    Isogenic ESC–derived neural cell types recapitulate patient-specific phenotypes. (A) CRISPR/Cas9 targeting scheme to introduce patient-specific deletion in WIBR#3 ESC line. (B) PCR assay shows untargeted control line and three heterozygous deletion lines. (C) Genomic DNA sequence of CRISPR target sites flanking the deletion. (D) Immunostaining for control, isogenic deletion, and iPSC lines after NPC differentiation. (E) Quantification of NPC fraction. Normalized data are shown as mean ± SEM (n = 4). (F) Comparative protein analysis of control, isogenic deletion, and iPSC lines upon neuronal differentiation. Arrows indicate three isoforms of SHANK3. (G) Quantification of immunoblots shown in (F). Data are represented as mean ± SEM (n = 3).

    Journal: Life Science Alliance

    Article Title: JIP2 haploinsufficiency contributes to neurodevelopmental abnormalities in human pluripotent stem cell–derived neural progenitors and cortical neurons

    doi: 10.26508/lsa.201800094

    Figure Lengend Snippet: Isogenic ESC–derived neural cell types recapitulate patient-specific phenotypes. (A) CRISPR/Cas9 targeting scheme to introduce patient-specific deletion in WIBR#3 ESC line. (B) PCR assay shows untargeted control line and three heterozygous deletion lines. (C) Genomic DNA sequence of CRISPR target sites flanking the deletion. (D) Immunostaining for control, isogenic deletion, and iPSC lines after NPC differentiation. (E) Quantification of NPC fraction. Normalized data are shown as mean ± SEM (n = 4). (F) Comparative protein analysis of control, isogenic deletion, and iPSC lines upon neuronal differentiation. Arrows indicate three isoforms of SHANK3. (G) Quantification of immunoblots shown in (F). Data are represented as mean ± SEM (n = 3).

    Article Snippet: For qRT–PCR, cDNA was synthesized using SuperScript III First Strand Synthesis Kit (#18080051; Thermo Fisher Scientific).

    Techniques: Derivative Assay, CRISPR, Introduce, Polymerase Chain Reaction, Sequencing, Immunostaining, Western Blot

    PAX6-GFP reporter lines allow specific purification of human PSC-derived neural progenitors. (A) Sequencing of the second allele of three independent clones. (B) FACS purification of three independent clones plus negative control after NPC differentiation. (C) Comparative gene expression profiles: global versus differentially expressed genes. (D) Comparative transcript profiling of a subset of NPC markers. (E) qRT–PCR validation of a subset of NPC markers. Error bars represent SEM. (F) Gene expression upstream and across the 22q13 deletion. Data are represented as log2 RPM values.

    Journal: Life Science Alliance

    Article Title: JIP2 haploinsufficiency contributes to neurodevelopmental abnormalities in human pluripotent stem cell–derived neural progenitors and cortical neurons

    doi: 10.26508/lsa.201800094

    Figure Lengend Snippet: PAX6-GFP reporter lines allow specific purification of human PSC-derived neural progenitors. (A) Sequencing of the second allele of three independent clones. (B) FACS purification of three independent clones plus negative control after NPC differentiation. (C) Comparative gene expression profiles: global versus differentially expressed genes. (D) Comparative transcript profiling of a subset of NPC markers. (E) qRT–PCR validation of a subset of NPC markers. Error bars represent SEM. (F) Gene expression upstream and across the 22q13 deletion. Data are represented as log2 RPM values.

    Article Snippet: For qRT–PCR, cDNA was synthesized using SuperScript III First Strand Synthesis Kit (#18080051; Thermo Fisher Scientific).

    Techniques: Purification, Derivative Assay, Sequencing, FACS, Clone Assay, Negative Control, Expressing, Quantitative RT-PCR

    Pharmacological activation of the NRP1 recues JNK expression in maturing patient neurons. (A) Mechanistic scheme of the semaphorin pathway in neurons. (B) Comparative transcript analysis (RNAseq) of key factors of semaphorin pathway and JNK target genes. (C) Protein analysis of mature neurons derived from control and two independent patient iPSC lines. (D) Quantification of immunoblots shown in (C). Data are represented as mean ± SEM (n = 3). (E) Transcript analysis (qRT–PCR) of key factors of the semaphorin pathway upon treatment with recombinant SEMA3A. Data are represented as mean ± SEM (two biological replicates including three technical replicates each). Two-tailed paired t test, * P

    Journal: Life Science Alliance

    Article Title: JIP2 haploinsufficiency contributes to neurodevelopmental abnormalities in human pluripotent stem cell–derived neural progenitors and cortical neurons

    doi: 10.26508/lsa.201800094

    Figure Lengend Snippet: Pharmacological activation of the NRP1 recues JNK expression in maturing patient neurons. (A) Mechanistic scheme of the semaphorin pathway in neurons. (B) Comparative transcript analysis (RNAseq) of key factors of semaphorin pathway and JNK target genes. (C) Protein analysis of mature neurons derived from control and two independent patient iPSC lines. (D) Quantification of immunoblots shown in (C). Data are represented as mean ± SEM (n = 3). (E) Transcript analysis (qRT–PCR) of key factors of the semaphorin pathway upon treatment with recombinant SEMA3A. Data are represented as mean ± SEM (two biological replicates including three technical replicates each). Two-tailed paired t test, * P

    Article Snippet: For qRT–PCR, cDNA was synthesized using SuperScript III First Strand Synthesis Kit (#18080051; Thermo Fisher Scientific).

    Techniques: Activation Assay, Expressing, Derivative Assay, Western Blot, Quantitative RT-PCR, Recombinant, Two Tailed Test