qpcr rna  (New England Biolabs)


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    Name:
    NEBNext Poly A mRNA Magnetic Isolation Module
    Description:
    NEBNext Poly A mRNA Magnetic Isolation Module 96 rxns
    Catalog Number:
    e7490l
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    242
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    96 rxns
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    mRNA Purification Kits
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    New England Biolabs qpcr rna
    NEBNext Poly A mRNA Magnetic Isolation Module
    NEBNext Poly A mRNA Magnetic Isolation Module 96 rxns
    https://www.bioz.com/result/qpcr rna/product/New England Biolabs
    Average 92 stars, based on 976 article reviews
    Price from $9.99 to $1999.99
    qpcr rna - by Bioz Stars, 2021-02
    92/100 stars

    Images

    1) Product Images from "The stronger downregulation of in vitro and in vivo innate antiviral responses by a very virulent strain of infectious bursal disease virus (IBDV), compared to a classical strain, is mediated, in part, by the VP4 protein"

    Article Title: The stronger downregulation of in vitro and in vivo innate antiviral responses by a very virulent strain of infectious bursal disease virus (IBDV), compared to a classical strain, is mediated, in part, by the VP4 protein

    Journal: bioRxiv

    doi: 10.1101/2019.12.17.879437

    The expression of type I IFN and pro-inflammatory genes was significantly reduced in B cells infected with strain UK661 compared to strain F52/70 in vitro . DT40 Cells were infected at an MOI of 0.1 with either the UK661 or F52/70 IBDV strains, or mock-infected with media alone and RNA was extracted from the cells at the indicated time points post-infection. RNA was reverse transcribed and amplified by qPCR using specific primer sets. The CT values were normalised to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (A). The log 2 fold change in host-cell gene expression was also determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (B-G). Data subsequently passed a Shapiro-Wilk normality test before being analysed by a one-way ANOVA and a Tukey’s multiple comparison test (*P
    Figure Legend Snippet: The expression of type I IFN and pro-inflammatory genes was significantly reduced in B cells infected with strain UK661 compared to strain F52/70 in vitro . DT40 Cells were infected at an MOI of 0.1 with either the UK661 or F52/70 IBDV strains, or mock-infected with media alone and RNA was extracted from the cells at the indicated time points post-infection. RNA was reverse transcribed and amplified by qPCR using specific primer sets. The CT values were normalised to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (A). The log 2 fold change in host-cell gene expression was also determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (B-G). Data subsequently passed a Shapiro-Wilk normality test before being analysed by a one-way ANOVA and a Tukey’s multiple comparison test (*P

    Techniques Used: Expressing, Infection, In Vitro, Amplification, Real-time Polymerase Chain Reaction

    The UK661 strain was more virulent than the F52/70 strain, but both strains replicated to the same peak titre in vivo . Birds were checked twice daily by two independent observers for clinical signs and a Kaplan Meier survival curve plotted of mock- (black), F52/70- (pink) and UK661- (grey) inoculated birds that reached their humane end points (clinical score of 11) (A). Clinical signs were quantified by a scoring system and divided into mild (1-7) and moderate (8-11). Each bird was assigned a clinical score at the indicated time points post-infection (B). Six birds per group were humanely culled at 24 and 48 hours post-infection (hpi), one F52/70 and three UK661-infected birds reached their humane end-points at 54 hpi and the remaining birds were culled at 72 hpi. The bursa of Fabricius was harvested at necropsy and the log 10 fold change in viral RNA copies/g tissue determined by RT-qPCR (C). The infectious titre was determined by titration onto DT40 cells in the method described by Reed Muench. Virus titres were expressed as log 10 TCID 50 /g of tissue (D). The horizontal lines are the mean values. Data passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P
    Figure Legend Snippet: The UK661 strain was more virulent than the F52/70 strain, but both strains replicated to the same peak titre in vivo . Birds were checked twice daily by two independent observers for clinical signs and a Kaplan Meier survival curve plotted of mock- (black), F52/70- (pink) and UK661- (grey) inoculated birds that reached their humane end points (clinical score of 11) (A). Clinical signs were quantified by a scoring system and divided into mild (1-7) and moderate (8-11). Each bird was assigned a clinical score at the indicated time points post-infection (B). Six birds per group were humanely culled at 24 and 48 hours post-infection (hpi), one F52/70 and three UK661-infected birds reached their humane end-points at 54 hpi and the remaining birds were culled at 72 hpi. The bursa of Fabricius was harvested at necropsy and the log 10 fold change in viral RNA copies/g tissue determined by RT-qPCR (C). The infectious titre was determined by titration onto DT40 cells in the method described by Reed Muench. Virus titres were expressed as log 10 TCID 50 /g of tissue (D). The horizontal lines are the mean values. Data passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P

    Techniques Used: In Vivo, Infection, Quantitative RT-PCR, Titration, Two Tailed Test

    The ability of the UK661 VP4 protein to antagonise type I IFN responses was reduced in the context of the whole virus in vitro . Birds were inoculated with 1.8×10 3 TCID 50 of the PBG98, PBG98-VP4 UK661 and PBG98-VP4 F52/70 viruses, and the bursa of Fabricius was harvested at necropsy from 6 birds per group at 2, 4 and 14 days post-inoculation. RNA was extracted prior to reverse transcription to cDNA and qPCR amplification with virus-specific primers. CT values were normalised to a housekeeping gene and expressed as log 10 fold change viral RNA relative to mock-infected samples as per the ΔΔCT method. The data passed a Shapiro-Wilk normality test before being analysed using a two-way ANOVA (not significant) (A). At 2 and 4 days post-inoculation, cDNA was amplified by qPCR for a panel genes: IFNα (B), IFNβ (C), Mx1 (D), IL-1β (E), and IL-8 (F). The CT values were normalised to the housekeeping gene RPLPO and expressed relative to mock-infected samples using the ΔΔCT method. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P
    Figure Legend Snippet: The ability of the UK661 VP4 protein to antagonise type I IFN responses was reduced in the context of the whole virus in vitro . Birds were inoculated with 1.8×10 3 TCID 50 of the PBG98, PBG98-VP4 UK661 and PBG98-VP4 F52/70 viruses, and the bursa of Fabricius was harvested at necropsy from 6 birds per group at 2, 4 and 14 days post-inoculation. RNA was extracted prior to reverse transcription to cDNA and qPCR amplification with virus-specific primers. CT values were normalised to a housekeeping gene and expressed as log 10 fold change viral RNA relative to mock-infected samples as per the ΔΔCT method. The data passed a Shapiro-Wilk normality test before being analysed using a two-way ANOVA (not significant) (A). At 2 and 4 days post-inoculation, cDNA was amplified by qPCR for a panel genes: IFNα (B), IFNβ (C), Mx1 (D), IL-1β (E), and IL-8 (F). The CT values were normalised to the housekeeping gene RPLPO and expressed relative to mock-infected samples using the ΔΔCT method. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P

    Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Amplification, Infection, Two Tailed Test

    The ability of the UK661 VP4 protein to antagonise type I IFN responses was reduced in the context of the whole virus in vitro . DF-1 cells were infected with PBG98, PBG98-VP4 UK661 and PBG98-VP4 F52/70 viruses at an MOI of 1, before RNA was extracted at the indicated time points post-infection and reverse transcribed. Virus specific primers were used to amplify the cDNA by quantitative PCR, the CT values were normalised to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression was determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. A Kruskal-Wallis test was performed with a Dunn’s multiple comparison test where no significant difference was found at any time point between the three viruses (A). A panel of genes, IFNα (B), IFNβ (C), Mx1 (D), IL-1β (E), and IL-8 (F), were amplified by quantitative PCR using specific primer sets for target genes, before the CT values were normalised to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P
    Figure Legend Snippet: The ability of the UK661 VP4 protein to antagonise type I IFN responses was reduced in the context of the whole virus in vitro . DF-1 cells were infected with PBG98, PBG98-VP4 UK661 and PBG98-VP4 F52/70 viruses at an MOI of 1, before RNA was extracted at the indicated time points post-infection and reverse transcribed. Virus specific primers were used to amplify the cDNA by quantitative PCR, the CT values were normalised to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression was determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. A Kruskal-Wallis test was performed with a Dunn’s multiple comparison test where no significant difference was found at any time point between the three viruses (A). A panel of genes, IFNα (B), IFNβ (C), Mx1 (D), IL-1β (E), and IL-8 (F), were amplified by quantitative PCR using specific primer sets for target genes, before the CT values were normalised to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P

    Techniques Used: In Vitro, Infection, Real-time Polymerase Chain Reaction, Expressing, Amplification, Two Tailed Test

    The expression of type I IFN and pro-inflammatory genes was significantly reduced in BF tissue harvested from birds infected with strain UK661 compared to strain F52/70 in vivo . The bursa of Fabricius was harvested from mock and infected birds at necropsy and RNA extracted. RNA was reverse transcribed and amplified by quantitative PCR using specific primer sets for target genes. The CT values were normalised to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted for individual birds. The horizontal lines are the mean values. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P
    Figure Legend Snippet: The expression of type I IFN and pro-inflammatory genes was significantly reduced in BF tissue harvested from birds infected with strain UK661 compared to strain F52/70 in vivo . The bursa of Fabricius was harvested from mock and infected birds at necropsy and RNA extracted. RNA was reverse transcribed and amplified by quantitative PCR using specific primer sets for target genes. The CT values were normalised to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted for individual birds. The horizontal lines are the mean values. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P

    Techniques Used: Expressing, Infection, In Vivo, Amplification, Real-time Polymerase Chain Reaction, Two Tailed Test

    2) Product Images from "CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti"

    Article Title: CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-58731-y

    RNA interference (RNAi) of CAPAr abolishes anti-diuretic activity of CAPA neuropeptide on adult female A. aegypti MTs. ( A ) Verification of significant knockdown ( > 75%) of CAPAr transcript in MTs of four-day old adult female A. aegypti by RNAi achieved through injection of dsCAPAr on day one post-eclosion. ( B ) Functional consequences of CAPAr knockdown demonstrating loss of anti-diuretic hormone activity by Aedae CAPA-1 against Drome DH 31 -stimulated fluid secretion by MTs. In ( A ), knockdown of CAPAr transcript was analyzed by one-tailed t-test (* denotes significant knockdown, p
    Figure Legend Snippet: RNA interference (RNAi) of CAPAr abolishes anti-diuretic activity of CAPA neuropeptide on adult female A. aegypti MTs. ( A ) Verification of significant knockdown ( > 75%) of CAPAr transcript in MTs of four-day old adult female A. aegypti by RNAi achieved through injection of dsCAPAr on day one post-eclosion. ( B ) Functional consequences of CAPAr knockdown demonstrating loss of anti-diuretic hormone activity by Aedae CAPA-1 against Drome DH 31 -stimulated fluid secretion by MTs. In ( A ), knockdown of CAPAr transcript was analyzed by one-tailed t-test (* denotes significant knockdown, p

    Techniques Used: Activity Assay, Injection, Functional Assay, One-tailed Test

    3) Product Images from "lncRNA CASC2 downregulation participates in rheumatoid arthritis, and CASC2 overexpression promotes the apoptosis of fibroblast-like synoviocytes by downregulating IL-17"

    Article Title: lncRNA CASC2 downregulation participates in rheumatoid arthritis, and CASC2 overexpression promotes the apoptosis of fibroblast-like synoviocytes by downregulating IL-17

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2020.11018

    lncRNA CASC2 is downregulated, while IL-17 is upregulated in the plasma of RA patients. (A) Relative expression of lncRNA CASC2 was normalized to the patients with the lowest expression level. qPCR results showed that, compared with the healthy controls, plasma levels of lncRNA CASC2 were significantly decreased in patients with RA. (B) In contrast, ELISA results showed that plasma IL-17 was upregulated in the plasma of RA patients when compared to that noted in the plasma of healthy controls (*P
    Figure Legend Snippet: lncRNA CASC2 is downregulated, while IL-17 is upregulated in the plasma of RA patients. (A) Relative expression of lncRNA CASC2 was normalized to the patients with the lowest expression level. qPCR results showed that, compared with the healthy controls, plasma levels of lncRNA CASC2 were significantly decreased in patients with RA. (B) In contrast, ELISA results showed that plasma IL-17 was upregulated in the plasma of RA patients when compared to that noted in the plasma of healthy controls (*P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    lncRNA CASC2 is a potential upstream inhibitor of IL-17 in HFLSs. (A) Overexpression of lncRNA CASC2 inhibited IL-17 expression in HFLSs, while (B) treatment with IL-17 did not significantly affect the expression of lncRNA CASC2 (*P
    Figure Legend Snippet: lncRNA CASC2 is a potential upstream inhibitor of IL-17 in HFLSs. (A) Overexpression of lncRNA CASC2 inhibited IL-17 expression in HFLSs, while (B) treatment with IL-17 did not significantly affect the expression of lncRNA CASC2 (*P

    Techniques Used: Over Expression, Expressing

    Altered plasma levels of lncRNA CASC2 and IL-17 differentiate RA patients from healthy controls. ROC curve analysis showed that altered plasma levels of (A) lncRNA CASC2 and (B) IL-17 were able to differentiate RA patients from healthy controls. CASC2, lncRNA cancer susceptibility candidate 2; IL-17, interleukin-17; RA, rheumatoid arthritis.
    Figure Legend Snippet: Altered plasma levels of lncRNA CASC2 and IL-17 differentiate RA patients from healthy controls. ROC curve analysis showed that altered plasma levels of (A) lncRNA CASC2 and (B) IL-17 were able to differentiate RA patients from healthy controls. CASC2, lncRNA cancer susceptibility candidate 2; IL-17, interleukin-17; RA, rheumatoid arthritis.

    Techniques Used:

    lncRNA CASC2 overexpression promotes while IL-17 inhibits the apoptosis of HFLSs. (A and B) Overexpression of lncRNA CASC2 was reached after transfection in HFLSs isolated from 2 RA patients. (C and D) Overexpression of lncRNA CASC2 led to a significant promotion of the percentage of apoptosis in HFLSs isolated from 2 RA patients. In contrast, treatment with IL-17 at a dose of 10 ng/ml significantly inhibited the apoptosis of HFLSs. In addition, IL-17 treatment attenuated the promoting effect of lncRNA CASC2 overexpression on cell apoptosis (*P
    Figure Legend Snippet: lncRNA CASC2 overexpression promotes while IL-17 inhibits the apoptosis of HFLSs. (A and B) Overexpression of lncRNA CASC2 was reached after transfection in HFLSs isolated from 2 RA patients. (C and D) Overexpression of lncRNA CASC2 led to a significant promotion of the percentage of apoptosis in HFLSs isolated from 2 RA patients. In contrast, treatment with IL-17 at a dose of 10 ng/ml significantly inhibited the apoptosis of HFLSs. In addition, IL-17 treatment attenuated the promoting effect of lncRNA CASC2 overexpression on cell apoptosis (*P

    Techniques Used: Over Expression, Transfection, Isolation

    Plasma levels of lncRNA CASC2 and IL-17 are significantly and inversely correlated in both RA patients and healthy controls. Relative expression of lncRNA CASC2 was normalized to the patients with the lowest expression level. Pearson's correlation coefficient showed that plasma levels of lncRNA CASC2 and IL-17 were significantly and inversely correlated in both (A) RA patients and (B) healthy controls. CASC2, lncRNA cancer susceptibility candidate 2; IL-17, interleukin-17; RA, rheumatoid arthritis.
    Figure Legend Snippet: Plasma levels of lncRNA CASC2 and IL-17 are significantly and inversely correlated in both RA patients and healthy controls. Relative expression of lncRNA CASC2 was normalized to the patients with the lowest expression level. Pearson's correlation coefficient showed that plasma levels of lncRNA CASC2 and IL-17 were significantly and inversely correlated in both (A) RA patients and (B) healthy controls. CASC2, lncRNA cancer susceptibility candidate 2; IL-17, interleukin-17; RA, rheumatoid arthritis.

    Techniques Used: Expressing

    4) Product Images from "Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump"

    Article Title: Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0243082

    Effect of chlorhexidine and resveratrol alone or in combination on expression of efflux pump and efflux pump regulator genes in A . baumannii strain L14. RT-qPCR assay of adeB (a), adeR (b), adeS (c), adeJ (d), adeG (e), abeS (f), and aceI (g) expression in the presence of MHB control, DMSO control, either chlorhexidine (8 mg/L) or resveratrol (128 mg/L) and in the combination of chlorhexidine with resveratrol. Relative number of transcripts of each gene was normalized to 16S rRNA expression in each condition and calculated using the 2 -ΔΔct method compared to the expression level in MHB control. The relative number of transcripts of each gene was plotted with error bars representing standard deviations. All experiments were performed in triplicate. p- values were calculated using ANOVA (#, p -value
    Figure Legend Snippet: Effect of chlorhexidine and resveratrol alone or in combination on expression of efflux pump and efflux pump regulator genes in A . baumannii strain L14. RT-qPCR assay of adeB (a), adeR (b), adeS (c), adeJ (d), adeG (e), abeS (f), and aceI (g) expression in the presence of MHB control, DMSO control, either chlorhexidine (8 mg/L) or resveratrol (128 mg/L) and in the combination of chlorhexidine with resveratrol. Relative number of transcripts of each gene was normalized to 16S rRNA expression in each condition and calculated using the 2 -ΔΔct method compared to the expression level in MHB control. The relative number of transcripts of each gene was plotted with error bars representing standard deviations. All experiments were performed in triplicate. p- values were calculated using ANOVA (#, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    5) Product Images from "DNA mismatch repair controls the host innate response and cell fate after influenza virus infection"

    Article Title: DNA mismatch repair controls the host innate response and cell fate after influenza virus infection

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0509-3

    Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.
    Figure Legend Snippet: Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.

    Techniques Used: Activity Assay, Expressing, Western Blot, Preserving, Infection, Transfection, Two Tailed Test

    6) Product Images from "An anti-diuretic hormone receptor in the human disease vector, Aedes aegypti: identification, expression analysis and functional deorphanization"

    Article Title: An anti-diuretic hormone receptor in the human disease vector, Aedes aegypti: identification, expression analysis and functional deorphanization

    Journal: bioRxiv

    doi: 10.1101/799833

    RNA interference (RNAi) of CAPAr abolishes anti-diuretic activity of CAPA neuropeptide on adult female A. aegypti MTs. (A) Verification of significant knockdown ( > 75%) of CAPAr transcript in MTs of four-day old adult female A. aegypti by RNAi achieved through injection of dsCAPAr on day one post-eclosion. (B) Functional consequences of CAPAr knockdown demonstrating loss of anti-diuretic hormone activity by Aedae CAPA-1 against Drome DH 31 -stimulated fluid secretion by MTs. In (A), knockdown of CAPAr transcript was analyzed by one-tailed t-test (* denotes significant knockdown, p
    Figure Legend Snippet: RNA interference (RNAi) of CAPAr abolishes anti-diuretic activity of CAPA neuropeptide on adult female A. aegypti MTs. (A) Verification of significant knockdown ( > 75%) of CAPAr transcript in MTs of four-day old adult female A. aegypti by RNAi achieved through injection of dsCAPAr on day one post-eclosion. (B) Functional consequences of CAPAr knockdown demonstrating loss of anti-diuretic hormone activity by Aedae CAPA-1 against Drome DH 31 -stimulated fluid secretion by MTs. In (A), knockdown of CAPAr transcript was analyzed by one-tailed t-test (* denotes significant knockdown, p

    Techniques Used: Activity Assay, Injection, Functional Assay, One-tailed Test

    7) Product Images from "Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food"

    Article Title: Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.00514

    RT-qPCR and qPCR of E. coli O157:H7 from 0, 4, 8, 24, and 72 h growth in BHI. (A) The growth curve of E. coli O157:H7 at 0, 4, 8, 24, 72 h in BHI. The initial concentration was 3-log CFU/mL. (B) RT-qPCR for RNA collected from five time points. (C) qPCR for 0–72 h RNA as the negative control (NC) for DNA contamination – no DNA contamination was found in those samples. (D) qPCR for DNA collected from five time points. (E) The melting curve analysis of RT-qPCR for 0–72 h RNA.
    Figure Legend Snippet: RT-qPCR and qPCR of E. coli O157:H7 from 0, 4, 8, 24, and 72 h growth in BHI. (A) The growth curve of E. coli O157:H7 at 0, 4, 8, 24, 72 h in BHI. The initial concentration was 3-log CFU/mL. (B) RT-qPCR for RNA collected from five time points. (C) qPCR for 0–72 h RNA as the negative control (NC) for DNA contamination – no DNA contamination was found in those samples. (D) qPCR for DNA collected from five time points. (E) The melting curve analysis of RT-qPCR for 0–72 h RNA.

    Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Concentration Assay, Negative Control

    Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex RT-PCR amplicon sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.
    Figure Legend Snippet: Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex RT-PCR amplicon sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.

    Techniques Used: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Generated

    8) Product Images from "Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies"

    Article Title: Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies

    Journal: Genes

    doi: 10.3390/genes10020079

    Functional annotation of the Tni-FNL ( Trichoplusia ni ), B. mori and D. melanogaster results comparison. The circos plot describes the shared cellular components, molecular functions and biological processes among the three species.
    Figure Legend Snippet: Functional annotation of the Tni-FNL ( Trichoplusia ni ), B. mori and D. melanogaster results comparison. The circos plot describes the shared cellular components, molecular functions and biological processes among the three species.

    Techniques Used: Functional Assay

    9) Product Images from "Towards deorphanizing G protein-coupled receptors of Schistosoma mansoni using the MALAR yeast two-hybrid system"

    Article Title: Towards deorphanizing G protein-coupled receptors of Schistosoma mansoni using the MALAR yeast two-hybrid system

    Journal: Parasitology

    doi: 10.1017/S0031182019001756

    Full-length expression of S. mansoni GPCRs in S. cerevisiae . Wild type or optimized CDSs of GPCRs were transformed into yeast strain AH109. RNA was isolated and transcribed into cDNA. Specific primer pairs were used to amplify corresponding GPCRs (numbers 1-7) by RT-PCR, and resulting amplicons were size-separated on a 1% agarose gel (M = marker, bp = base pair). The absence of reverse transcriptase (-RT) during cDNA synthesis or cDNA synthesis without template (H2O) served as negative controls. RNA isolated from S. mansoni served as a positive control for full-length transcripts. In each case, amplicons of the expected sizes were obtained.
    Figure Legend Snippet: Full-length expression of S. mansoni GPCRs in S. cerevisiae . Wild type or optimized CDSs of GPCRs were transformed into yeast strain AH109. RNA was isolated and transcribed into cDNA. Specific primer pairs were used to amplify corresponding GPCRs (numbers 1-7) by RT-PCR, and resulting amplicons were size-separated on a 1% agarose gel (M = marker, bp = base pair). The absence of reverse transcriptase (-RT) during cDNA synthesis or cDNA synthesis without template (H2O) served as negative controls. RNA isolated from S. mansoni served as a positive control for full-length transcripts. In each case, amplicons of the expected sizes were obtained.

    Techniques Used: Expressing, Transformation Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Positive Control

    10) Product Images from "Merkel Cell Polyomavirus Small Tumor Antigen Activates Matrix Metallopeptidase-9 Gene Expression for Cell Migration and Invasion"

    Article Title: Merkel Cell Polyomavirus Small Tumor Antigen Activates Matrix Metallopeptidase-9 Gene Expression for Cell Migration and Invasion

    Journal: bioRxiv

    doi: 10.1101/2020.03.02.974303

    MCV sT activates Matrix metalloproteinase 9 (MMP-9). (A) MCV sT expression results in upregulation of MMP-9 mRNA levels. Various cell lines (293, COS-7, MCC13 and U2OS) were transfected with either empty vector or MCV sT WT expressing plasmids to measure MMP-9 mRNA levels. After 48 h, total RNA was isolated and analyzed by RT-qPCR. (B) MCV sT upregulates MMP-9 transcription through the LSD. U2OS and MCC13 cells transfected with empty vector control, MCV sT WT and MCV sT LSDm expressing plasmids. Transcript levels of MMP-9 were analyzed using the comparative ΔΔCt method. (n = 3). Differences between means ( p value) were analyzed using a t-test with GraphPad Prism software. (C) MCV sT upregulates MMP-9 protein expression through the LSD. U2OS cells were transfected with empty vector, sT WT and sT LSDm expression plasmids. After 48 h, immunoblot analysis was performed to analyze expression of MMP-9, sT and α-tubulin (Ci). Densitometry quantification of immunoblots was carried out using the Image studio software and is shown as a fold change relative to the loading control α-tubulin (Cii). Data analyzed using three biological replicates per experiment (n = 3). (D) MCV sT reproducibly activates MMP-9 expression in MCC13.
    Figure Legend Snippet: MCV sT activates Matrix metalloproteinase 9 (MMP-9). (A) MCV sT expression results in upregulation of MMP-9 mRNA levels. Various cell lines (293, COS-7, MCC13 and U2OS) were transfected with either empty vector or MCV sT WT expressing plasmids to measure MMP-9 mRNA levels. After 48 h, total RNA was isolated and analyzed by RT-qPCR. (B) MCV sT upregulates MMP-9 transcription through the LSD. U2OS and MCC13 cells transfected with empty vector control, MCV sT WT and MCV sT LSDm expressing plasmids. Transcript levels of MMP-9 were analyzed using the comparative ΔΔCt method. (n = 3). Differences between means ( p value) were analyzed using a t-test with GraphPad Prism software. (C) MCV sT upregulates MMP-9 protein expression through the LSD. U2OS cells were transfected with empty vector, sT WT and sT LSDm expression plasmids. After 48 h, immunoblot analysis was performed to analyze expression of MMP-9, sT and α-tubulin (Ci). Densitometry quantification of immunoblots was carried out using the Image studio software and is shown as a fold change relative to the loading control α-tubulin (Cii). Data analyzed using three biological replicates per experiment (n = 3). (D) MCV sT reproducibly activates MMP-9 expression in MCC13.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR, Software, Western Blot

    MCV sT leads to differential expression of proteins associated with epithelial to mesenchymal transition (EMT). (A) Quantitative proteomics analysis illustrating differential expression of EMT associated proteins upon MCV sT expression. Proteins associated with cell adhesion and structural integrity of the extracellular matrix are downregulated upon MCV sT expression. While expression of proteins which encourage cell migration by reorganization of the actin network and microtubule destabilization are upregulated. (B) MCV sT regulates EMT-associated gene expression. MCC13 cells were transfected with control or MCV sT expression plasmids. While epithelial markers were downregulated, mesenchymal markers were significantly upregulated upon MCV sT expression. Cellular RNA was extracted using a trizol reagent and transcript levels were analyzed by RT-qPCR using the comparative ΔΔCt method (n = 3).
    Figure Legend Snippet: MCV sT leads to differential expression of proteins associated with epithelial to mesenchymal transition (EMT). (A) Quantitative proteomics analysis illustrating differential expression of EMT associated proteins upon MCV sT expression. Proteins associated with cell adhesion and structural integrity of the extracellular matrix are downregulated upon MCV sT expression. While expression of proteins which encourage cell migration by reorganization of the actin network and microtubule destabilization are upregulated. (B) MCV sT regulates EMT-associated gene expression. MCC13 cells were transfected with control or MCV sT expression plasmids. While epithelial markers were downregulated, mesenchymal markers were significantly upregulated upon MCV sT expression. Cellular RNA was extracted using a trizol reagent and transcript levels were analyzed by RT-qPCR using the comparative ΔΔCt method (n = 3).

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

    11) Product Images from "ETV7 represses a subset of interferon-stimulated genes that restrict influenza viruses"

    Article Title: ETV7 represses a subset of interferon-stimulated genes that restrict influenza viruses

    Journal: bioRxiv

    doi: 10.1101/851543

    ETV7 differentially regulates ISGs during the type I IFN response based on ISRE-related regulatory elements. A) Dendrogram of genes most differentially expressed in cells overexpressing either a control protein (mCherry) or ETV7 before and after IFN-α treatment (100 U/mL, 9 h) as measured using RNA sequencing. Three independent, biological replicates per condition. Red box highlights control samples, yellow box highlights ETV7-expressing samples, shading indicates IFN-stimulated samples. The box width indicates the linkage distance between samples before and after IFN, indicating control cells’ transcriptional profile is more diverged after IFN treatment compared to ETV7-expressing cells. B) Heat map displaying RNA levels of genes upregulated at least two-fold following IFN-α treatment (100 U/mL, 9 h) in control cells. Expression was normalized to control cells after IFN treatment (averaged across replicates). Yellow = downregulated, blue = upregulated. C,D) Motif counts in promoter regions (−1000 bp, +500 bp) for the genes most and least affected by ETV7 overexpression in the RNA sequencing results. ETS sites (GGAA) highlighted in yellow. P-values calculated using unpaired, two-tailed Mann-Whitney U tests (*p
    Figure Legend Snippet: ETV7 differentially regulates ISGs during the type I IFN response based on ISRE-related regulatory elements. A) Dendrogram of genes most differentially expressed in cells overexpressing either a control protein (mCherry) or ETV7 before and after IFN-α treatment (100 U/mL, 9 h) as measured using RNA sequencing. Three independent, biological replicates per condition. Red box highlights control samples, yellow box highlights ETV7-expressing samples, shading indicates IFN-stimulated samples. The box width indicates the linkage distance between samples before and after IFN, indicating control cells’ transcriptional profile is more diverged after IFN treatment compared to ETV7-expressing cells. B) Heat map displaying RNA levels of genes upregulated at least two-fold following IFN-α treatment (100 U/mL, 9 h) in control cells. Expression was normalized to control cells after IFN treatment (averaged across replicates). Yellow = downregulated, blue = upregulated. C,D) Motif counts in promoter regions (−1000 bp, +500 bp) for the genes most and least affected by ETV7 overexpression in the RNA sequencing results. ETS sites (GGAA) highlighted in yellow. P-values calculated using unpaired, two-tailed Mann-Whitney U tests (*p

    Techniques Used: RNA Sequencing Assay, Expressing, Over Expression, Two Tailed Test, MANN-WHITNEY

    ETV7 loss enhances expression of specific ISGs. A) ISG15 and ETV7 mRNA levels in A549 lung epithelial cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). B) Percentage of A549 cells expressing GFP from IFNrsp reporter after knockout of ETV7 and IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=3, statistical analysis represents p-values for both of the two ETV7 KO sgRNAs compared to a non-targeting control). C) mRNA levels of ETV7 in non-targeting control and ETV7 KO A549 cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). D,E) Representative genes were chosen from the groups D) most affected by ETV7 (Group I) and E) least affected (Groups II and III) in the RNA sequencing analysis ( Fig. 4B ). Each gene’s potential ISRE sequences (ETS sites highlighted in yellow) are shown, along with its mRNA levels in control and ETV7 KO cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). For all panels: Data shown are representative of two independent experiments. P-values calculated using unpaired, two-tailed Student’s t-tests (*p
    Figure Legend Snippet: ETV7 loss enhances expression of specific ISGs. A) ISG15 and ETV7 mRNA levels in A549 lung epithelial cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). B) Percentage of A549 cells expressing GFP from IFNrsp reporter after knockout of ETV7 and IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=3, statistical analysis represents p-values for both of the two ETV7 KO sgRNAs compared to a non-targeting control). C) mRNA levels of ETV7 in non-targeting control and ETV7 KO A549 cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). D,E) Representative genes were chosen from the groups D) most affected by ETV7 (Group I) and E) least affected (Groups II and III) in the RNA sequencing analysis ( Fig. 4B ). Each gene’s potential ISRE sequences (ETS sites highlighted in yellow) are shown, along with its mRNA levels in control and ETV7 KO cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). For all panels: Data shown are representative of two independent experiments. P-values calculated using unpaired, two-tailed Student’s t-tests (*p

    Techniques Used: Expressing, Knock-Out, RNA Sequencing Assay, Two Tailed Test

    12) Product Images from "Ascorbate sensitizes human osteosarcoma cells to the cytostatic effects of cisplatin, et al. Ascorbate sensitizes human osteosarcoma cells to the cytostatic effects of cisplatin"

    Article Title: Ascorbate sensitizes human osteosarcoma cells to the cytostatic effects of cisplatin, et al. Ascorbate sensitizes human osteosarcoma cells to the cytostatic effects of cisplatin

    Journal: Pharmacology Research & Perspectives

    doi: 10.1002/prp2.632

    Ascorbate enhances the effect of cisplatin in osteosarcoma cells. A‐C, U2OS cells (1500 cells) were treated with cisplatin (0‐100 µmol/L) (A), ascorbate (0‐100 µmol/L) (B), and cisplatin (0‐100 µmol/L) plus ascorbate (1, 10, and 30 µmol/L) (C) for 96 h. D‐F, 143B cells (1,500 cells) were treated with cisplatin (0‐100 µmol/L) (D), ascorbate (0‐100 µmol/L) (E), and cisplatin (0‐100 µmol/L) plus ascorbate (1, 10, and 30 µmol/L) (F) for 96 h. G‐I, Nonmalignant human lung fibroblast, IMR‐90 cells (1,500 cells), were treated with cisplatin (0‐100 µmol/L) (G), ascorbate (0‐100 µmol/L) (H), and cisplatin (0‐100 µmol/L) plus ascorbate (1, 10, and 30 µmol/L) (I) for 96 h. Cell viability was quantified by the cell viability assay. The data represent the mean ± SD of triplicate samples from three independent experiments. * P
    Figure Legend Snippet: Ascorbate enhances the effect of cisplatin in osteosarcoma cells. A‐C, U2OS cells (1500 cells) were treated with cisplatin (0‐100 µmol/L) (A), ascorbate (0‐100 µmol/L) (B), and cisplatin (0‐100 µmol/L) plus ascorbate (1, 10, and 30 µmol/L) (C) for 96 h. D‐F, 143B cells (1,500 cells) were treated with cisplatin (0‐100 µmol/L) (D), ascorbate (0‐100 µmol/L) (E), and cisplatin (0‐100 µmol/L) plus ascorbate (1, 10, and 30 µmol/L) (F) for 96 h. G‐I, Nonmalignant human lung fibroblast, IMR‐90 cells (1,500 cells), were treated with cisplatin (0‐100 µmol/L) (G), ascorbate (0‐100 µmol/L) (H), and cisplatin (0‐100 µmol/L) plus ascorbate (1, 10, and 30 µmol/L) (I) for 96 h. Cell viability was quantified by the cell viability assay. The data represent the mean ± SD of triplicate samples from three independent experiments. * P

    Techniques Used: Viability Assay

    Ascorbate treatment sensitizes cisplatin‐resistant U2OS cells to cisplatin treatment. A, RNA sequence analysis revealed that ribosome biogenesis and mitochondrial respiration were enhanced in CisLS‐U2OS cells. B, Upregulation of Myc target genes in CisLS‐U2OS cells. C, Mitochondrial (basal respiration, ATP production, maximal respiration, and spare capacity) (left) and glycolytic functions (right) were measured by a flux analyzer in parental and CisLS‐U2OS cells (n = 3). D, Parental and CisLS‐U2OS cells (1x10 4 cells) were treated with cisplatin (0‐100 µmol/L) or cisplatin plus ascorbate (10 µmol/L) for 96 h and the number of live cells were measured by cell proliferation assay (n = 6). Data are presented as the mean ± SD from three independent experiments. * P
    Figure Legend Snippet: Ascorbate treatment sensitizes cisplatin‐resistant U2OS cells to cisplatin treatment. A, RNA sequence analysis revealed that ribosome biogenesis and mitochondrial respiration were enhanced in CisLS‐U2OS cells. B, Upregulation of Myc target genes in CisLS‐U2OS cells. C, Mitochondrial (basal respiration, ATP production, maximal respiration, and spare capacity) (left) and glycolytic functions (right) were measured by a flux analyzer in parental and CisLS‐U2OS cells (n = 3). D, Parental and CisLS‐U2OS cells (1x10 4 cells) were treated with cisplatin (0‐100 µmol/L) or cisplatin plus ascorbate (10 µmol/L) for 96 h and the number of live cells were measured by cell proliferation assay (n = 6). Data are presented as the mean ± SD from three independent experiments. * P

    Techniques Used: Sequencing, Proliferation Assay

    Ascorbate and cisplatin treatment alter mitochondrial respiration and glucose metabolism of U2OS cells. A, U2OS cells (1 × 10 4 cells) were treated with cisplatin (10 µmol/L), ascorbate (10 µmol/L), or cisplatin plus ascorbate for 96 h and the glycolytic functions (basal and compensatory glycolysis) were measured by a flux analyzer. Kinetics of the ECAR response of U2OS cells to ROT/AA (0.5 µmol/L) or 2‐DG (50 mmol/L) was measured by a flux analyzer. B, U2OS cells (1 × 10 4 cells) were treated with cisplatin (10 µmol/L), ascorbate (10 µmol/L), or both for 96 h and the mitochondrial functions (basal respiration, ATP production, maximal respiration, and spare capacity) were measured by a flux analyzer. Kinetics of the OCR response of U2OS cells to oligomycin (1.0 µmol/L), FCCP (1.0 µmol/L), or ROT/AA (0.5 µmol/L) was measured by a flux analyzer. The data represent the mean ± SD of triplicate samples from three independent experiments. * P
    Figure Legend Snippet: Ascorbate and cisplatin treatment alter mitochondrial respiration and glucose metabolism of U2OS cells. A, U2OS cells (1 × 10 4 cells) were treated with cisplatin (10 µmol/L), ascorbate (10 µmol/L), or cisplatin plus ascorbate for 96 h and the glycolytic functions (basal and compensatory glycolysis) were measured by a flux analyzer. Kinetics of the ECAR response of U2OS cells to ROT/AA (0.5 µmol/L) or 2‐DG (50 mmol/L) was measured by a flux analyzer. B, U2OS cells (1 × 10 4 cells) were treated with cisplatin (10 µmol/L), ascorbate (10 µmol/L), or both for 96 h and the mitochondrial functions (basal respiration, ATP production, maximal respiration, and spare capacity) were measured by a flux analyzer. Kinetics of the OCR response of U2OS cells to oligomycin (1.0 µmol/L), FCCP (1.0 µmol/L), or ROT/AA (0.5 µmol/L) was measured by a flux analyzer. The data represent the mean ± SD of triplicate samples from three independent experiments. * P

    Techniques Used:

    Ascorbate enhances ROS production in osteosarcoma cells. A, ROS levels in U2OS cells treated with cisplatin (0‐100 µmol/L) and ascorbate (10 µmol/L) for 96 h as measured by flow cytometry. Intracellular ROS levels were determined by measuring the mean fluorescence intensity (MFI) of DHE‐positive cells. MFI in the treated cells was expressed relative to MFI of the untreated cells (set at 1). B, ROS levels in U2OS cells measured by flow cytometry, 24, 48, and 96 h after treatment with cisplatin (0‐30 µmol/L) and ascorbate (10 µmol/L). MFI in the treated cells was expressed relative to MFI of the untreated cells (set at 1). C, U2OS cells were treated with cisplatin (10 µmol/L) and/or ascorbate (10 µmol/L) in the presence or absence of ROS scavenger NAC (2 mmol/L), 96 h after treatment and the number of γH2AX dots was counted. D, Immunostaining of γH2AX (green) and DAPI (blue). The data represent the mean ± SD of triplicate samples from three independent experiments. * P
    Figure Legend Snippet: Ascorbate enhances ROS production in osteosarcoma cells. A, ROS levels in U2OS cells treated with cisplatin (0‐100 µmol/L) and ascorbate (10 µmol/L) for 96 h as measured by flow cytometry. Intracellular ROS levels were determined by measuring the mean fluorescence intensity (MFI) of DHE‐positive cells. MFI in the treated cells was expressed relative to MFI of the untreated cells (set at 1). B, ROS levels in U2OS cells measured by flow cytometry, 24, 48, and 96 h after treatment with cisplatin (0‐30 µmol/L) and ascorbate (10 µmol/L). MFI in the treated cells was expressed relative to MFI of the untreated cells (set at 1). C, U2OS cells were treated with cisplatin (10 µmol/L) and/or ascorbate (10 µmol/L) in the presence or absence of ROS scavenger NAC (2 mmol/L), 96 h after treatment and the number of γH2AX dots was counted. D, Immunostaining of γH2AX (green) and DAPI (blue). The data represent the mean ± SD of triplicate samples from three independent experiments. * P

    Techniques Used: Flow Cytometry, Fluorescence, Immunostaining

    Cisplatin and ascorbate treatment affect the expression of rate‐limiting enzymes in glucose metabolism. qRT‐PCR analysis of the expression of hexokinase 2 (HK2), monocarboxylate transporter 4 (MCT4), glucose‐6‐phosphate dehydrogenase (G6PD), and lactate dehydrogenase A (LDHA) in U2OS cells treated with cisplatin (10 µmol/L), ascorbate (10 µmol/L), or both for 96 h in the presence or absence of NAC (2 mmol/L). HRPT1, control. Data are presented as the mean ± SD of triplicate samples from three independent experiments. * P
    Figure Legend Snippet: Cisplatin and ascorbate treatment affect the expression of rate‐limiting enzymes in glucose metabolism. qRT‐PCR analysis of the expression of hexokinase 2 (HK2), monocarboxylate transporter 4 (MCT4), glucose‐6‐phosphate dehydrogenase (G6PD), and lactate dehydrogenase A (LDHA) in U2OS cells treated with cisplatin (10 µmol/L), ascorbate (10 µmol/L), or both for 96 h in the presence or absence of NAC (2 mmol/L). HRPT1, control. Data are presented as the mean ± SD of triplicate samples from three independent experiments. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    13) Product Images from "Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump"

    Article Title: Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0243082

    Accumulation of ethidium bromide in A . baumannii strain AC152, AC154, L14, and L21. The fluorescence representing ethidium bromide accumulation was measured in the presence of glucose as an energy source every 40 seconds for 1600 seconds. Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.
    Figure Legend Snippet: Accumulation of ethidium bromide in A . baumannii strain AC152, AC154, L14, and L21. The fluorescence representing ethidium bromide accumulation was measured in the presence of glucose as an energy source every 40 seconds for 1600 seconds. Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.

    Techniques Used: Fluorescence

    Time-kill curves of resveratrol and chlorhexidine against A . baumannii . Time-kill curves of resveratrol 128 mg/L in combination with 0.25x MIC of chlorhexidine (4 or 8 mg/L) against A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21(d). Mean values of viable cells were plotted with error bars representing standard deviations. All experiments were performed in triplicate and the detection limit of the viable cells is 10 2 CFU/mL.
    Figure Legend Snippet: Time-kill curves of resveratrol and chlorhexidine against A . baumannii . Time-kill curves of resveratrol 128 mg/L in combination with 0.25x MIC of chlorhexidine (4 or 8 mg/L) against A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21(d). Mean values of viable cells were plotted with error bars representing standard deviations. All experiments were performed in triplicate and the detection limit of the viable cells is 10 2 CFU/mL.

    Techniques Used:

    Effect of resveratrol in the presence of chlorhexidine on ethidium bromide accumulation in A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21 (d). The fluorescence of ethidium bromide was measured in the presence of glucose with (filled triangles) or without (open circles) chlorhexidine (8 mg/L) and after addition of CCCP (open squares) or resveratrol (open triangles). Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.
    Figure Legend Snippet: Effect of resveratrol in the presence of chlorhexidine on ethidium bromide accumulation in A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21 (d). The fluorescence of ethidium bromide was measured in the presence of glucose with (filled triangles) or without (open circles) chlorhexidine (8 mg/L) and after addition of CCCP (open squares) or resveratrol (open triangles). Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.

    Techniques Used: Fluorescence

    Effect of chlorhexidine and resveratrol alone or in combination on expression of efflux pump and efflux pump regulator genes in A . baumannii strain L14. RT-qPCR assay of adeB (a), adeR (b), adeS (c), adeJ (d), adeG (e), abeS (f), and aceI (g) expression in the presence of MHB control, DMSO control, either chlorhexidine (8 mg/L) or resveratrol (128 mg/L) and in the combination of chlorhexidine with resveratrol. Relative number of transcripts of each gene was normalized to 16S rRNA expression in each condition and calculated using the 2 -ΔΔct method compared to the expression level in MHB control. The relative number of transcripts of each gene was plotted with error bars representing standard deviations. All experiments were performed in triplicate. p- values were calculated using ANOVA (#, p -value
    Figure Legend Snippet: Effect of chlorhexidine and resveratrol alone or in combination on expression of efflux pump and efflux pump regulator genes in A . baumannii strain L14. RT-qPCR assay of adeB (a), adeR (b), adeS (c), adeJ (d), adeG (e), abeS (f), and aceI (g) expression in the presence of MHB control, DMSO control, either chlorhexidine (8 mg/L) or resveratrol (128 mg/L) and in the combination of chlorhexidine with resveratrol. Relative number of transcripts of each gene was normalized to 16S rRNA expression in each condition and calculated using the 2 -ΔΔct method compared to the expression level in MHB control. The relative number of transcripts of each gene was plotted with error bars representing standard deviations. All experiments were performed in triplicate. p- values were calculated using ANOVA (#, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of resveratrol on ethidium bromide accumulation in A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21 (d). The fluorescence of ethidium bromide was measured in the presence of glucose and after addition of CCCP (filled squares), resveratrol (filled triangles), or control with no addition of any proton coupler agent (fill circles). Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.
    Figure Legend Snippet: Effect of resveratrol on ethidium bromide accumulation in A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21 (d). The fluorescence of ethidium bromide was measured in the presence of glucose and after addition of CCCP (filled squares), resveratrol (filled triangles), or control with no addition of any proton coupler agent (fill circles). Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.

    Techniques Used: Fluorescence

    14) Product Images from "Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti"

    Article Title: Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2019.00266

    Distinct localization of LGR1 immunoreactivity in late-staged spermatids of adult A. aegypti testes. (A,C,E,G) Testes were stained with anti-LGR1 (green), (B,C) anti-gamma tubulin (red) as a marker for the centriole adjunct ( Dallai et al., 2016 ), (F,G) MitoTracker (red) to detect mitochondrial derivatives in flagella, and nuclei (blue). (C) LGR1 immunoreactivity co-localizes with gamma-tubulin at the centriole adjunct. (D) Transmission electron microscopy (TEM) image showing the centriole adjunct (arrow) is positioned between the nucleus (n) and flagella (f) of spermatids. (G) LGR1 immunoreactivity is not found associated with the mitochondrial derivatives in the flagella of late-staged spermatids, comprised of (H) two mitochondrial derivatives (m) and an axoneme (a). Early staged spermatids (est), late-staged spermatids (Lst), cyst cell nuclei ( ∗ ). Scale bars: 10 μm in panels A–C , 5 μm in panel D , 20 μm in panels E–G and 200 nm in panel H .
    Figure Legend Snippet: Distinct localization of LGR1 immunoreactivity in late-staged spermatids of adult A. aegypti testes. (A,C,E,G) Testes were stained with anti-LGR1 (green), (B,C) anti-gamma tubulin (red) as a marker for the centriole adjunct ( Dallai et al., 2016 ), (F,G) MitoTracker (red) to detect mitochondrial derivatives in flagella, and nuclei (blue). (C) LGR1 immunoreactivity co-localizes with gamma-tubulin at the centriole adjunct. (D) Transmission electron microscopy (TEM) image showing the centriole adjunct (arrow) is positioned between the nucleus (n) and flagella (f) of spermatids. (G) LGR1 immunoreactivity is not found associated with the mitochondrial derivatives in the flagella of late-staged spermatids, comprised of (H) two mitochondrial derivatives (m) and an axoneme (a). Early staged spermatids (est), late-staged spermatids (Lst), cyst cell nuclei ( ∗ ). Scale bars: 10 μm in panels A–C , 5 μm in panel D , 20 μm in panels E–G and 200 nm in panel H .

    Techniques Used: Staining, Marker, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

    LGR1 knockdown efficiency in adult A. aegypti . Second instar larvae were fed with Escherichia coli expressing LGR1 dsRNA or control dsRNA (empty L4440 vector). After feeding, LGR1 transcript knockdown efficiency was determined in panel (A) whole 4th-instar larvae and one-day old adults by quantitative RT-PCR. LGR1 immunoreactivity was examined in control and LGR1 knockdown treatments within (B–F) the testes of virgin one-day adult males (LGR1, green; nuclei, blue). (A) LGR1 transcript levels of 4th instar larvae and newly emerged adults fed with LGR1 dsRNA were significantly reduced compared to control dsRNA-fed mosquitoes. (B) Quantification of LGR1 immunoreactivity from control and dsLGR1-treated larvae confirms downregulation. Representative confocal images demonstrate reduced LGR1 immunofluorescence in cysts (dotted outline) containing late-staged spermatids (Lst) when comparing (C,E) control and (D,F) dsLGR1-treated mosquitoes. All data are presented as mean ± SEM ( n = 3 in panel A , n = 11–13 in panel B ) with asterisks representing significant differences between control and dsLGR1-treated mosquitoes as determined using an unpaired t -test: ∗ P
    Figure Legend Snippet: LGR1 knockdown efficiency in adult A. aegypti . Second instar larvae were fed with Escherichia coli expressing LGR1 dsRNA or control dsRNA (empty L4440 vector). After feeding, LGR1 transcript knockdown efficiency was determined in panel (A) whole 4th-instar larvae and one-day old adults by quantitative RT-PCR. LGR1 immunoreactivity was examined in control and LGR1 knockdown treatments within (B–F) the testes of virgin one-day adult males (LGR1, green; nuclei, blue). (A) LGR1 transcript levels of 4th instar larvae and newly emerged adults fed with LGR1 dsRNA were significantly reduced compared to control dsRNA-fed mosquitoes. (B) Quantification of LGR1 immunoreactivity from control and dsLGR1-treated larvae confirms downregulation. Representative confocal images demonstrate reduced LGR1 immunofluorescence in cysts (dotted outline) containing late-staged spermatids (Lst) when comparing (C,E) control and (D,F) dsLGR1-treated mosquitoes. All data are presented as mean ± SEM ( n = 3 in panel A , n = 11–13 in panel B ) with asterisks representing significant differences between control and dsLGR1-treated mosquitoes as determined using an unpaired t -test: ∗ P

    Techniques Used: Expressing, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence

    15) Product Images from "fosA3 overexpression with transporter mutations mediates high-level of fosfomycin resistance and silence of fosA3 in fosfomycin-susceptible Klebsiella pneumoniae producing carbapenemase clinical isolates"

    Article Title: fosA3 overexpression with transporter mutations mediates high-level of fosfomycin resistance and silence of fosA3 in fosfomycin-susceptible Klebsiella pneumoniae producing carbapenemase clinical isolates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0237474

    Relative fosA5 expression levels of IMP-producing K . pneumoniae . qRT-PCR assay of fosA5 expression was performed in K . pneumoniae isolate KP35 and KP71. The relative number of transcripts of fosA5 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP35. p -values were calculated using unpaired t-test (*, p -value
    Figure Legend Snippet: Relative fosA5 expression levels of IMP-producing K . pneumoniae . qRT-PCR assay of fosA5 expression was performed in K . pneumoniae isolate KP35 and KP71. The relative number of transcripts of fosA5 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP35. p -values were calculated using unpaired t-test (*, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    Relative fosA5 and fosA3 expression levels of NDM and OXA-48-coproducing K . pneumoniae . qRT-PCR assay of fosA5 (a) and fosA3 (b) expression was performed in K . pneumoniae isolate KP5, KP4, KP6, KP15, KP18, KP19, and KP7. The relative number of transcripts of fosA5 and fosA3 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP5. p -values were calculated using unpaired t-test (*, p -value
    Figure Legend Snippet: Relative fosA5 and fosA3 expression levels of NDM and OXA-48-coproducing K . pneumoniae . qRT-PCR assay of fosA5 (a) and fosA3 (b) expression was performed in K . pneumoniae isolate KP5, KP4, KP6, KP15, KP18, KP19, and KP7. The relative number of transcripts of fosA5 and fosA3 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP5. p -values were calculated using unpaired t-test (*, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    Relative fosA5 and fosA3 expression levels of OXA-48-producing K . pneumoniae . qRT-PCR assay of fosA5 (a) and fosA3 (b, c) expression was performed in K . pneumoniae isolate KP27, KP11, KP44, and KP51. The relative number of transcripts of fosA5 and fosA3 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP27 or KP44. p -values were calculated using unpaired t-test (*, p -value
    Figure Legend Snippet: Relative fosA5 and fosA3 expression levels of OXA-48-producing K . pneumoniae . qRT-PCR assay of fosA5 (a) and fosA3 (b, c) expression was performed in K . pneumoniae isolate KP27, KP11, KP44, and KP51. The relative number of transcripts of fosA5 and fosA3 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP27 or KP44. p -values were calculated using unpaired t-test (*, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    Relative fosA5 and fosA3 expression levels of NDM-producing K . pneumoniae . qRT-PCR assay of fosA5 (a) and fosA3 (b) expression was performed in K . pneumoniae isolate KP23 and KP58. The relative number of transcripts of fosA5 and fosA3 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP23. p -values were calculated using unpaired t-test (*, p -value
    Figure Legend Snippet: Relative fosA5 and fosA3 expression levels of NDM-producing K . pneumoniae . qRT-PCR assay of fosA5 (a) and fosA3 (b) expression was performed in K . pneumoniae isolate KP23 and KP58. The relative number of transcripts of fosA5 and fosA3 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP23. p -values were calculated using unpaired t-test (*, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    16) Product Images from "Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food"

    Article Title: Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.00514

    RT-qPCR and qPCR of E. coli O157:H7 from 0, 4, 8, 24, and 72 h growth in BHI. (A) The growth curve of E. coli O157:H7 at 0, 4, 8, 24, 72 h in BHI. The initial concentration was 3-log CFU/mL. (B) RT-qPCR for RNA collected from five time points. (C) qPCR for 0–72 h RNA as the negative control (NC) for DNA contamination – no DNA contamination was found in those samples. (D) qPCR for DNA collected from five time points. (E) The melting curve analysis of RT-qPCR for 0–72 h RNA.
    Figure Legend Snippet: RT-qPCR and qPCR of E. coli O157:H7 from 0, 4, 8, 24, and 72 h growth in BHI. (A) The growth curve of E. coli O157:H7 at 0, 4, 8, 24, 72 h in BHI. The initial concentration was 3-log CFU/mL. (B) RT-qPCR for RNA collected from five time points. (C) qPCR for 0–72 h RNA as the negative control (NC) for DNA contamination – no DNA contamination was found in those samples. (D) qPCR for DNA collected from five time points. (E) The melting curve analysis of RT-qPCR for 0–72 h RNA.

    Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Concentration Assay, Negative Control

    17) Product Images from "Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food"

    Article Title: Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.00514

    Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex RT-PCR amplicon sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.
    Figure Legend Snippet: Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex RT-PCR amplicon sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.

    Techniques Used: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Generated

    18) Product Images from "Towards deorphanizing G protein-coupled receptors of Schistosoma mansoni using the MALAR yeast two-hybrid system"

    Article Title: Towards deorphanizing G protein-coupled receptors of Schistosoma mansoni using the MALAR yeast two-hybrid system

    Journal: Parasitology

    doi: 10.1017/S0031182019001756

    Full-length expression of S. mansoni GPCRs in S. cerevisiae . Wild type or optimized CDSs of GPCRs were transformed into yeast strain AH109. RNA was isolated and transcribed into cDNA. Specific primer pairs were used to amplify corresponding GPCRs (numbers 1-7) by RT-PCR, and resulting amplicons were size-separated on a 1% agarose gel (M = marker, bp = base pair). The absence of reverse transcriptase (-RT) during cDNA synthesis or cDNA synthesis without template (H2O) served as negative controls. RNA isolated from S. mansoni served as a positive control for full-length transcripts. In each case, amplicons of the expected sizes were obtained.
    Figure Legend Snippet: Full-length expression of S. mansoni GPCRs in S. cerevisiae . Wild type or optimized CDSs of GPCRs were transformed into yeast strain AH109. RNA was isolated and transcribed into cDNA. Specific primer pairs were used to amplify corresponding GPCRs (numbers 1-7) by RT-PCR, and resulting amplicons were size-separated on a 1% agarose gel (M = marker, bp = base pair). The absence of reverse transcriptase (-RT) during cDNA synthesis or cDNA synthesis without template (H2O) served as negative controls. RNA isolated from S. mansoni served as a positive control for full-length transcripts. In each case, amplicons of the expected sizes were obtained.

    Techniques Used: Expressing, Transformation Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Positive Control

    19) Product Images from "Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump"

    Article Title: Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0243082

    Time-kill curves of resveratrol and chlorhexidine against A . baumannii . Time-kill curves of resveratrol 128 mg/L in combination with 0.25x MIC of chlorhexidine (4 or 8 mg/L) against A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21(d). Mean values of viable cells were plotted with error bars representing standard deviations. All experiments were performed in triplicate and the detection limit of the viable cells is 10 2 CFU/mL.
    Figure Legend Snippet: Time-kill curves of resveratrol and chlorhexidine against A . baumannii . Time-kill curves of resveratrol 128 mg/L in combination with 0.25x MIC of chlorhexidine (4 or 8 mg/L) against A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21(d). Mean values of viable cells were plotted with error bars representing standard deviations. All experiments were performed in triplicate and the detection limit of the viable cells is 10 2 CFU/mL.

    Techniques Used:

    Effect of resveratrol in the presence of chlorhexidine on ethidium bromide accumulation in A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21 (d). The fluorescence of ethidium bromide was measured in the presence of glucose with (filled triangles) or without (open circles) chlorhexidine (8 mg/L) and after addition of CCCP (open squares) or resveratrol (open triangles). Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.
    Figure Legend Snippet: Effect of resveratrol in the presence of chlorhexidine on ethidium bromide accumulation in A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21 (d). The fluorescence of ethidium bromide was measured in the presence of glucose with (filled triangles) or without (open circles) chlorhexidine (8 mg/L) and after addition of CCCP (open squares) or resveratrol (open triangles). Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.

    Techniques Used: Fluorescence

    Effect of chlorhexidine and resveratrol alone or in combination on expression of efflux pump and efflux pump regulator genes in A . baumannii strain L14. RT-qPCR assay of adeB (a), adeR (b), adeS (c), adeJ (d), adeG (e), abeS (f), and aceI (g) expression in the presence of MHB control, DMSO control, either chlorhexidine (8 mg/L) or resveratrol (128 mg/L) and in the combination of chlorhexidine with resveratrol. Relative number of transcripts of each gene was normalized to 16S rRNA expression in each condition and calculated using the 2 -ΔΔct method compared to the expression level in MHB control. The relative number of transcripts of each gene was plotted with error bars representing standard deviations. All experiments were performed in triplicate. p- values were calculated using ANOVA (#, p -value
    Figure Legend Snippet: Effect of chlorhexidine and resveratrol alone or in combination on expression of efflux pump and efflux pump regulator genes in A . baumannii strain L14. RT-qPCR assay of adeB (a), adeR (b), adeS (c), adeJ (d), adeG (e), abeS (f), and aceI (g) expression in the presence of MHB control, DMSO control, either chlorhexidine (8 mg/L) or resveratrol (128 mg/L) and in the combination of chlorhexidine with resveratrol. Relative number of transcripts of each gene was normalized to 16S rRNA expression in each condition and calculated using the 2 -ΔΔct method compared to the expression level in MHB control. The relative number of transcripts of each gene was plotted with error bars representing standard deviations. All experiments were performed in triplicate. p- values were calculated using ANOVA (#, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of resveratrol on ethidium bromide accumulation in A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21 (d). The fluorescence of ethidium bromide was measured in the presence of glucose and after addition of CCCP (filled squares), resveratrol (filled triangles), or control with no addition of any proton coupler agent (fill circles). Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.
    Figure Legend Snippet: Effect of resveratrol on ethidium bromide accumulation in A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21 (d). The fluorescence of ethidium bromide was measured in the presence of glucose and after addition of CCCP (filled squares), resveratrol (filled triangles), or control with no addition of any proton coupler agent (fill circles). Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.

    Techniques Used: Fluorescence

    20) Product Images from "An anti-diuretic hormone receptor in the human disease vector, Aedes aegypti: identification, expression analysis and functional deorphanization"

    Article Title: An anti-diuretic hormone receptor in the human disease vector, Aedes aegypti: identification, expression analysis and functional deorphanization

    Journal: bioRxiv

    doi: 10.1101/799833

    Expression analysis of CAPAr transcript in the mosquito, A. aegypti . (A) Ontogenic expression profile of CAPAr transcript over post-embryonic stages of the A. aegypti mosquito shown relative to transcript levels in 1 st instar larvae. (B) Spatial expression is analyzed in various tissues/organs from four-day old adult females, with transcript abundance shown relative to levels in the male midgut. (C) Cell-specific expression of CAPAr mRNA in principal cells (arrows) of MTs from adult female A. aegypti detected using an anti-sense probe, with no detection in the stellate cells (arrowheads). (D) No signal was detected in preparations hybridized with control CAPAr sense probe. All images acquired using identical microscope settings; scale bars in C-D are 100μm.
    Figure Legend Snippet: Expression analysis of CAPAr transcript in the mosquito, A. aegypti . (A) Ontogenic expression profile of CAPAr transcript over post-embryonic stages of the A. aegypti mosquito shown relative to transcript levels in 1 st instar larvae. (B) Spatial expression is analyzed in various tissues/organs from four-day old adult females, with transcript abundance shown relative to levels in the male midgut. (C) Cell-specific expression of CAPAr mRNA in principal cells (arrows) of MTs from adult female A. aegypti detected using an anti-sense probe, with no detection in the stellate cells (arrowheads). (D) No signal was detected in preparations hybridized with control CAPAr sense probe. All images acquired using identical microscope settings; scale bars in C-D are 100μm.

    Techniques Used: Expressing, Microscopy

    Expression analysis of CAPA neuropeptide (anti-diuretic hormone) transcript in different regions of the nervous system relative to whole adult (A) male and (B) female A. aegypti mosquitoes. Different letters denote bars that are significantly different from one another as determined by one-way ANOVA and Tukey’s multiple comparison post-hoc test (p
    Figure Legend Snippet: Expression analysis of CAPA neuropeptide (anti-diuretic hormone) transcript in different regions of the nervous system relative to whole adult (A) male and (B) female A. aegypti mosquitoes. Different letters denote bars that are significantly different from one another as determined by one-way ANOVA and Tukey’s multiple comparison post-hoc test (p

    Techniques Used: Expressing

    21) Product Images from "A novel isoform of ACE2 is expressed in human nasal and bronchial respiratory epithelia and is upregulated in response to RNA respiratory virus infection"

    Article Title: A novel isoform of ACE2 is expressed in human nasal and bronchial respiratory epithelia and is upregulated in response to RNA respiratory virus infection

    Journal: bioRxiv

    doi: 10.1101/2020.07.31.230870

    Long ACE2 is expressed at lower levels in bronchial brushings from severe asthmatic donors and RV-induced expression of long ACE2 is reduced in bronchial ALI cultures from severe asthmatic donors. 7a. Bronchial epithelial brushes from healthy controls (n=13) or severe asthmatic (n=11) donors were harvested and RNA extracted for analysis of ACE2 transcript expression by transcript-specific RT-qPCR. Data were analysed using nonparametric Mann-Whitney test. 7b. Bronchial epithelial cells from healthy (n=11) or severe asthmatic (n=7) donors were grown at ALI and infected with rhinovirus (RV16) (MOI of 1) or mock-infected using a UV-irradiated control. After 24h, induction of ACE2 transcripts was assessed by RT-qPCR with transcript-specific primers in duplicate. The fold-induction of each transcript by RV16 was quantified using the ddCt method using UV-RV exposed cells as control. Activation of anti-viral response in RV16-infected ALI cultures was demonstrated by detection of IL29/IL28 in basolateral supernatants by ELISA (healthy n=14, severe n=8). Data were analysed using Student’s t-test.
    Figure Legend Snippet: Long ACE2 is expressed at lower levels in bronchial brushings from severe asthmatic donors and RV-induced expression of long ACE2 is reduced in bronchial ALI cultures from severe asthmatic donors. 7a. Bronchial epithelial brushes from healthy controls (n=13) or severe asthmatic (n=11) donors were harvested and RNA extracted for analysis of ACE2 transcript expression by transcript-specific RT-qPCR. Data were analysed using nonparametric Mann-Whitney test. 7b. Bronchial epithelial cells from healthy (n=11) or severe asthmatic (n=7) donors were grown at ALI and infected with rhinovirus (RV16) (MOI of 1) or mock-infected using a UV-irradiated control. After 24h, induction of ACE2 transcripts was assessed by RT-qPCR with transcript-specific primers in duplicate. The fold-induction of each transcript by RV16 was quantified using the ddCt method using UV-RV exposed cells as control. Activation of anti-viral response in RV16-infected ALI cultures was demonstrated by detection of IL29/IL28 in basolateral supernatants by ELISA (healthy n=14, severe n=8). Data were analysed using Student’s t-test.

    Techniques Used: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Infection, Irradiation, Activation Assay, Enzyme-linked Immunosorbent Assay

    Short ACE2 is upregulated in response to IFN-beta and rhinovirus (RV16) infection but not SARS-CoV-2 infection 6a. Undifferentiated primary bronchial epithelial cell (PBEC) monolayer cultures (N=3) (top) or in vitro differentiated (ALI) PBEC cultures (N=3) (bottom) were treated with IFN-beta (100 or 1000 IU/ml) for 24h and ACE2 transcripts (left panel) and induction of IFN-response genes (MX1 and CXCL10) (right panel) were measured by RT-qPCR. Data were analysed using Students t-test. 6b. in vitro differentiated (ALI) nasal epithelia cells (NEC) (n=11) (left panel) or in vitro differentiated (ALI) bronchial epithelia cells (BEC) (n=11) (right panel) were infected with rhinovirus (RV16) (MOI of 1) or mock-infected using a UV-irradiated control (UV-RV16). Nasal cells were collected from 3 female, 8 male patients with a mean age of 45.31+/-3.23 (SEM). After 24h, induction of ACE2 isoform expression was assessed by RT-qPCR with transcript-specific primers. Data were analysed using non-parametric Wilcoxon test. 6c. BCi-NS1.1 cells were grown at ALI and then infected on the apical side for 1 hour with 100,000 pfu of SARS-Cov-2 strain nCoV/Victoria/1/2020 obtained from Public Health England (PHE), UK. Cells were harvested in QIAzol at 1h post infection and at 72h, RNA extracted and quantitative RT-qPCR performed to detect SARS-CoV-2 using 2019-nCoV_N1 primers and the housekeeping genes HPRT, 18S and RNAse P using the dCt method. 1 hour and 72 hours after infection induction of ACE2 transcript expression was assessed by RT-qPCR with transcript-specific primers (left). SARS-CoV-1 infection was confirmed by CoV-N1 RT-qPCR 1 and 72 hours after infection (right). Data were analysed using non-parametric Wilcoxon test. N=4.
    Figure Legend Snippet: Short ACE2 is upregulated in response to IFN-beta and rhinovirus (RV16) infection but not SARS-CoV-2 infection 6a. Undifferentiated primary bronchial epithelial cell (PBEC) monolayer cultures (N=3) (top) or in vitro differentiated (ALI) PBEC cultures (N=3) (bottom) were treated with IFN-beta (100 or 1000 IU/ml) for 24h and ACE2 transcripts (left panel) and induction of IFN-response genes (MX1 and CXCL10) (right panel) were measured by RT-qPCR. Data were analysed using Students t-test. 6b. in vitro differentiated (ALI) nasal epithelia cells (NEC) (n=11) (left panel) or in vitro differentiated (ALI) bronchial epithelia cells (BEC) (n=11) (right panel) were infected with rhinovirus (RV16) (MOI of 1) or mock-infected using a UV-irradiated control (UV-RV16). Nasal cells were collected from 3 female, 8 male patients with a mean age of 45.31+/-3.23 (SEM). After 24h, induction of ACE2 isoform expression was assessed by RT-qPCR with transcript-specific primers. Data were analysed using non-parametric Wilcoxon test. 6c. BCi-NS1.1 cells were grown at ALI and then infected on the apical side for 1 hour with 100,000 pfu of SARS-Cov-2 strain nCoV/Victoria/1/2020 obtained from Public Health England (PHE), UK. Cells were harvested in QIAzol at 1h post infection and at 72h, RNA extracted and quantitative RT-qPCR performed to detect SARS-CoV-2 using 2019-nCoV_N1 primers and the housekeeping genes HPRT, 18S and RNAse P using the dCt method. 1 hour and 72 hours after infection induction of ACE2 transcript expression was assessed by RT-qPCR with transcript-specific primers (left). SARS-CoV-1 infection was confirmed by CoV-N1 RT-qPCR 1 and 72 hours after infection (right). Data were analysed using non-parametric Wilcoxon test. N=4.

    Techniques Used: Infection, In Vitro, Quantitative RT-PCR, Irradiation, Expressing

    22) Product Images from "The Stronger Downregulation of in vitro and in vivo Innate Antiviral Responses by a Very Virulent Strain of Infectious Bursal Disease Virus (IBDV), Compared to a Classical Strain, Is Mediated, in Part, by the VP4 Protein"

    Article Title: The Stronger Downregulation of in vitro and in vivo Innate Antiviral Responses by a Very Virulent Strain of Infectious Bursal Disease Virus (IBDV), Compared to a Classical Strain, Is Mediated, in Part, by the VP4 Protein

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2020.00315

    The PBG98-VP4 UK661 virus inhibits the induction of IFNα and IL-8 to a greater extent than the PBG98-VP4 F52/70 virus in vitro . DF-1 cells were infected with PBG98, PBG98-VP4 UK661 , and PBG98-VP4 F52/70 viruses at an MOI of 1, before RNA was extracted at the indicated time points post-infection and reverse transcribed. Virus specific primers were used to amplify the cDNA by quantitative (q)PCR, the CT values were normalized to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression was determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. A one-way ANOVA and a Tukey's multiple comparison test was performed where no significant difference was found at any time point between the three viruses (A) . A panel of genes, IFNα (B) , IFNβ (C) , Mx1 (D) , IL-1β (E) , and IL-8 (F) , were amplified by qPCR using specific primer sets for target genes, before the CT values were normalized to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before being analyzed by a one-way ANOVA and a Tukey's multiple comparison test ( * p
    Figure Legend Snippet: The PBG98-VP4 UK661 virus inhibits the induction of IFNα and IL-8 to a greater extent than the PBG98-VP4 F52/70 virus in vitro . DF-1 cells were infected with PBG98, PBG98-VP4 UK661 , and PBG98-VP4 F52/70 viruses at an MOI of 1, before RNA was extracted at the indicated time points post-infection and reverse transcribed. Virus specific primers were used to amplify the cDNA by quantitative (q)PCR, the CT values were normalized to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression was determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. A one-way ANOVA and a Tukey's multiple comparison test was performed where no significant difference was found at any time point between the three viruses (A) . A panel of genes, IFNα (B) , IFNβ (C) , Mx1 (D) , IL-1β (E) , and IL-8 (F) , were amplified by qPCR using specific primer sets for target genes, before the CT values were normalized to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before being analyzed by a one-way ANOVA and a Tukey's multiple comparison test ( * p

    Techniques Used: In Vitro, Infection, Expressing, Amplification, Real-time Polymerase Chain Reaction

    The expression of type I IFN and pro-inflammatory genes was significantly reduced in BF tissue harvested from birds infected with strain UK661 compared to strain F52/70 in vivo . The bursa of Fabricius was harvested from mock and infected birds at necropsy and RNA extracted. RNA was reverse transcribed and amplified by quantitative PCR using specific primer sets for target genes. IFNα (A) , IFNβ (B) , Mx1 (C) , IL-1β (D) , IL-6 (E) , and IL8 (F) . The CT values were normalized to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted for individual birds. The horizontal lines are the mean values. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before being analyzed by a one-way ANOVA and a Tukey's multiple comparison test ( * p
    Figure Legend Snippet: The expression of type I IFN and pro-inflammatory genes was significantly reduced in BF tissue harvested from birds infected with strain UK661 compared to strain F52/70 in vivo . The bursa of Fabricius was harvested from mock and infected birds at necropsy and RNA extracted. RNA was reverse transcribed and amplified by quantitative PCR using specific primer sets for target genes. IFNα (A) , IFNβ (B) , Mx1 (C) , IL-1β (D) , IL-6 (E) , and IL8 (F) . The CT values were normalized to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted for individual birds. The horizontal lines are the mean values. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before being analyzed by a one-way ANOVA and a Tukey's multiple comparison test ( * p

    Techniques Used: Expressing, Infection, In Vivo, Amplification, Real-time Polymerase Chain Reaction

    The expression of type I IFN and pro-inflammatory genes was significantly reduced in B cells infected with strain UK661 compared to strain F52/70 in vitro . DT40 Cells were infected at an MOI of 0.1 with either the UK661 or F52/70 IBDV strains, or mock-infected with media alone and RNA was extracted from the cells at the indicated time points post-infection. RNA was reverse transcribed and amplified by qPCR using specific primer sets. The CT values were normalized to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (A) . The log 2 fold change in host-cell gene expression was also determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (B–G) . Data subsequently passed a Shapiro-Wilk normality test before being analyzed by a one-way ANOVA and a Tukey's multiple comparison test ( * P
    Figure Legend Snippet: The expression of type I IFN and pro-inflammatory genes was significantly reduced in B cells infected with strain UK661 compared to strain F52/70 in vitro . DT40 Cells were infected at an MOI of 0.1 with either the UK661 or F52/70 IBDV strains, or mock-infected with media alone and RNA was extracted from the cells at the indicated time points post-infection. RNA was reverse transcribed and amplified by qPCR using specific primer sets. The CT values were normalized to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (A) . The log 2 fold change in host-cell gene expression was also determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (B–G) . Data subsequently passed a Shapiro-Wilk normality test before being analyzed by a one-way ANOVA and a Tukey's multiple comparison test ( * P

    Techniques Used: Expressing, Infection, In Vitro, Amplification, Real-time Polymerase Chain Reaction

    The PBG98-VP4 UK661 virus inhibits the induction of IFNα to a greater extent than the PBG98-VP4 F52/70 virus in vivo . Birds were inoculated with 1.8 × 10 3 TCID 50 of the PBG98, PBG98-VP4 UK661 , and PBG98-VP4 F52/70 viruses, and the bursa of Fabricius was harvested at necropsy from 6 birds per group at 2, 4, and 14 days post-inoculation. RNA was extracted prior to reverse transcription to cDNA and qPCR amplification with virus-specific primers. CT values were normalized to a housekeeping gene and expressed as log 10 fold change viral RNA relative to mock-infected samples as per the ΔΔCT method. The data passed a Shapiro-Wilk normality test before being analyzed by a one-way ANOVA and a Tukey's multiple comparison test (not significant) (A) . At 2 and 4 days post-inoculation, cDNA was amplified by qPCR for a panel genes: IFNα (B) , IFNβ (C) , Mx1 (D) , IL-1β (E) , and IL-8 (F) . The CT values were normalized to the housekeeping gene RPLPO and expressed relative to mock-infected samples using the ΔΔCT method. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before being analyzed by a one-way ANOVA and a Tukey's multiple comparison test ( * p
    Figure Legend Snippet: The PBG98-VP4 UK661 virus inhibits the induction of IFNα to a greater extent than the PBG98-VP4 F52/70 virus in vivo . Birds were inoculated with 1.8 × 10 3 TCID 50 of the PBG98, PBG98-VP4 UK661 , and PBG98-VP4 F52/70 viruses, and the bursa of Fabricius was harvested at necropsy from 6 birds per group at 2, 4, and 14 days post-inoculation. RNA was extracted prior to reverse transcription to cDNA and qPCR amplification with virus-specific primers. CT values were normalized to a housekeeping gene and expressed as log 10 fold change viral RNA relative to mock-infected samples as per the ΔΔCT method. The data passed a Shapiro-Wilk normality test before being analyzed by a one-way ANOVA and a Tukey's multiple comparison test (not significant) (A) . At 2 and 4 days post-inoculation, cDNA was amplified by qPCR for a panel genes: IFNα (B) , IFNβ (C) , Mx1 (D) , IL-1β (E) , and IL-8 (F) . The CT values were normalized to the housekeeping gene RPLPO and expressed relative to mock-infected samples using the ΔΔCT method. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before being analyzed by a one-way ANOVA and a Tukey's multiple comparison test ( * p

    Techniques Used: In Vivo, Real-time Polymerase Chain Reaction, Amplification, Infection

    The UK661 strain was more virulent than the F52/70 strain, but both strains replicated to the same peak titer in vivo . Birds were checked twice daily by two independent observers for clinical signs and a Kaplan Meier survival curve plotted of mock- (black), F52/70- (pink), and UK661- (gray) inoculated birds that reached their humane end points (clinical score of 11) (A) . Clinical signs were quantified by a scoring system and divided into mild (1-7) and moderate (8-11). Each bird was assigned a clinical score at the indicated time points post-infection (B) . Six birds per group were humanely culled at 24 and 48 h post-infection (hpi), one F52/70 and three UK661-infected birds reached their humane end-points at 54 hpi and the remaining birds were culled at 72 hpi. The bursa of Fabricius was harvested at necropsy and the log 10 fold change in viral RNA copies/g tissue determined by RT-qPCR (C) . The infectious titer was determined by titration onto DT40 cells in the method described by Reed and Muench. Virus titers were expressed as log 10 TCID 50 /g of tissue (D) . The horizontal lines are the mean values. Data passed a Shapiro-Wilk normality test before being analyzed by a one-way ANOVA and a Tukey's multiple comparison test ( ** p
    Figure Legend Snippet: The UK661 strain was more virulent than the F52/70 strain, but both strains replicated to the same peak titer in vivo . Birds were checked twice daily by two independent observers for clinical signs and a Kaplan Meier survival curve plotted of mock- (black), F52/70- (pink), and UK661- (gray) inoculated birds that reached their humane end points (clinical score of 11) (A) . Clinical signs were quantified by a scoring system and divided into mild (1-7) and moderate (8-11). Each bird was assigned a clinical score at the indicated time points post-infection (B) . Six birds per group were humanely culled at 24 and 48 h post-infection (hpi), one F52/70 and three UK661-infected birds reached their humane end-points at 54 hpi and the remaining birds were culled at 72 hpi. The bursa of Fabricius was harvested at necropsy and the log 10 fold change in viral RNA copies/g tissue determined by RT-qPCR (C) . The infectious titer was determined by titration onto DT40 cells in the method described by Reed and Muench. Virus titers were expressed as log 10 TCID 50 /g of tissue (D) . The horizontal lines are the mean values. Data passed a Shapiro-Wilk normality test before being analyzed by a one-way ANOVA and a Tukey's multiple comparison test ( ** p

    Techniques Used: In Vivo, Infection, Quantitative RT-PCR, Titration

    23) Product Images from "KV7 Channel Expression and Function Within Rat Mesenteric Endothelial Cells"

    Article Title: KV7 Channel Expression and Function Within Rat Mesenteric Endothelial Cells

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2020.598779

    Expression profile of Kcnq genes within isolated rat mesenteric vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). RT-qPCR analysis of relative expression of Kcnq1-5 in isolated VSMCs and ECs (A) . Cell specific markers Acta2 and Pecam , for VSMCs and ECs respectively, were measured to determine sample purity (B,C) . Values expressed as mean ± SEM of (2 –ΔCq ) of ΔCq values generated from appropriate housekeeper genes ( n = 3). Statistical significance is defined as * P
    Figure Legend Snippet: Expression profile of Kcnq genes within isolated rat mesenteric vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). RT-qPCR analysis of relative expression of Kcnq1-5 in isolated VSMCs and ECs (A) . Cell specific markers Acta2 and Pecam , for VSMCs and ECs respectively, were measured to determine sample purity (B,C) . Values expressed as mean ± SEM of (2 –ΔCq ) of ΔCq values generated from appropriate housekeeper genes ( n = 3). Statistical significance is defined as * P

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Generated

    24) Product Images from "KV7 Channel Expression and Function Within Rat Mesenteric Endothelial Cells"

    Article Title: KV7 Channel Expression and Function Within Rat Mesenteric Endothelial Cells

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2020.598779

    Expression profile of Kcnq genes within isolated rat mesenteric vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). RT-qPCR analysis of relative expression of Kcnq1-5 in isolated VSMCs and ECs (A) . Cell specific markers Acta2 and Pecam , for VSMCs and ECs respectively, were measured to determine sample purity (B,C) . Values expressed as mean ± SEM of (2 –ΔCq ) of ΔCq values generated from appropriate housekeeper genes ( n = 3). Statistical significance is defined as * P
    Figure Legend Snippet: Expression profile of Kcnq genes within isolated rat mesenteric vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). RT-qPCR analysis of relative expression of Kcnq1-5 in isolated VSMCs and ECs (A) . Cell specific markers Acta2 and Pecam , for VSMCs and ECs respectively, were measured to determine sample purity (B,C) . Values expressed as mean ± SEM of (2 –ΔCq ) of ΔCq values generated from appropriate housekeeper genes ( n = 3). Statistical significance is defined as * P

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Generated

    25) Product Images from "fosA3 overexpression with transporter mutations mediates high-level of fosfomycin resistance and silence of fosA3 in fosfomycin-susceptible Klebsiella pneumoniae producing carbapenemase clinical isolates"

    Article Title: fosA3 overexpression with transporter mutations mediates high-level of fosfomycin resistance and silence of fosA3 in fosfomycin-susceptible Klebsiella pneumoniae producing carbapenemase clinical isolates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0237474

    Relative fosA5 expression levels of IMP-producing K . pneumoniae . qRT-PCR assay of fosA5 expression was performed in K . pneumoniae isolate KP35 and KP71. The relative number of transcripts of fosA5 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP35. p -values were calculated using unpaired t-test (*, p -value
    Figure Legend Snippet: Relative fosA5 expression levels of IMP-producing K . pneumoniae . qRT-PCR assay of fosA5 expression was performed in K . pneumoniae isolate KP35 and KP71. The relative number of transcripts of fosA5 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP35. p -values were calculated using unpaired t-test (*, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    Distribution of Fraction Inhibitory Concentration Index (FICI) of K . pneumoniae isolates. The FICIs of K . pneumoniae were plotted with different carbapenemase and fosA genes and categorized by synergism (FICI≤0.5), no interaction (0.5
    Figure Legend Snippet: Distribution of Fraction Inhibitory Concentration Index (FICI) of K . pneumoniae isolates. The FICIs of K . pneumoniae were plotted with different carbapenemase and fosA genes and categorized by synergism (FICI≤0.5), no interaction (0.5

    Techniques Used: Concentration Assay

    Relative fosA5 and fosA3 expression levels of NDM and OXA-48-coproducing K . pneumoniae . qRT-PCR assay of fosA5 (a) and fosA3 (b) expression was performed in K . pneumoniae isolate KP5, KP4, KP6, KP15, KP18, KP19, and KP7. The relative number of transcripts of fosA5 and fosA3 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP5. p -values were calculated using unpaired t-test (*, p -value
    Figure Legend Snippet: Relative fosA5 and fosA3 expression levels of NDM and OXA-48-coproducing K . pneumoniae . qRT-PCR assay of fosA5 (a) and fosA3 (b) expression was performed in K . pneumoniae isolate KP5, KP4, KP6, KP15, KP18, KP19, and KP7. The relative number of transcripts of fosA5 and fosA3 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP5. p -values were calculated using unpaired t-test (*, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    Relative fosA5 and fosA3 expression levels of OXA-48-producing K . pneumoniae . qRT-PCR assay of fosA5 (a) and fosA3 (b, c) expression was performed in K . pneumoniae isolate KP27, KP11, KP44, and KP51. The relative number of transcripts of fosA5 and fosA3 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP27 or KP44. p -values were calculated using unpaired t-test (*, p -value
    Figure Legend Snippet: Relative fosA5 and fosA3 expression levels of OXA-48-producing K . pneumoniae . qRT-PCR assay of fosA5 (a) and fosA3 (b, c) expression was performed in K . pneumoniae isolate KP27, KP11, KP44, and KP51. The relative number of transcripts of fosA5 and fosA3 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP27 or KP44. p -values were calculated using unpaired t-test (*, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    Growth of K . pneumoniae isolates in M9 minimal medium supplemented with G3P or G6P as a carbon source. The ability of K . pneumoniae growth was determined by measurement of the OD 600nm of the cell suspension, normalized to the OD 600nm of M9 supplemented with G6P or G3P without bacterial inoculation and compared to growth ability of E . coli ATCC 25922.
    Figure Legend Snippet: Growth of K . pneumoniae isolates in M9 minimal medium supplemented with G3P or G6P as a carbon source. The ability of K . pneumoniae growth was determined by measurement of the OD 600nm of the cell suspension, normalized to the OD 600nm of M9 supplemented with G6P or G3P without bacterial inoculation and compared to growth ability of E . coli ATCC 25922.

    Techniques Used:

    Relative fosA5 and fosA3 expression levels of NDM-producing K . pneumoniae . qRT-PCR assay of fosA5 (a) and fosA3 (b) expression was performed in K . pneumoniae isolate KP23 and KP58. The relative number of transcripts of fosA5 and fosA3 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP23. p -values were calculated using unpaired t-test (*, p -value
    Figure Legend Snippet: Relative fosA5 and fosA3 expression levels of NDM-producing K . pneumoniae . qRT-PCR assay of fosA5 (a) and fosA3 (b) expression was performed in K . pneumoniae isolate KP23 and KP58. The relative number of transcripts of fosA5 and fosA3 were normalized to 16S rRNA expression and calculated using the 2 -ΔΔct method compared to the expression of fosfomycin-susceptible K . pneumoniae isolate KP23. p -values were calculated using unpaired t-test (*, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    26) Product Images from "Phosphorylation of JIP4 at S730 presents anti-viral properties against influenza A virus infection"

    Article Title: Phosphorylation of JIP4 at S730 presents anti-viral properties against influenza A virus infection

    Journal: bioRxiv

    doi: 10.1101/2021.01.22.427772

    JIP4 interferes with IAV replication prior to viral protein expression A549 cells were transfected with either, control siRNAs or siRNAs targeting JIP4, and infected with PR8 (MOI 5). Cells were lysed every 2 h. Lysates were submitted to Western blot assay and incubated with antibodies against viral proteins PB1 and NS1, as well as against JIP4 for silencing confirmation. Detection of tubulin served as loading control (A) . A549 cells were transfected with either, empty vector (EV) or plasmids encoding JIP4, and infected with PR8 (MOI 5). Cells were lysed every 2 h. Lysates were submitted to Western Blot assay and incubated with antibodies against viral proteins PB1, NS1 and M1, as well as against JIP4 for overexpression confirmation. Detection of tubulin was used as loading control (B) . For analyses of viral mRNA expression, A549 cells were transfected with either siRNAs control or targeting JIP4 and infected with PR8 (MOI 5). Samples were collected every 2 h and progressed for qRT-PCR analyses. Viral mRNAs were normalized over 2 h control group. SPAG9 gene was normalized to MOCK control. Results were statistically analyzed by two-way ANOVA test (C) . **p
    Figure Legend Snippet: JIP4 interferes with IAV replication prior to viral protein expression A549 cells were transfected with either, control siRNAs or siRNAs targeting JIP4, and infected with PR8 (MOI 5). Cells were lysed every 2 h. Lysates were submitted to Western blot assay and incubated with antibodies against viral proteins PB1 and NS1, as well as against JIP4 for silencing confirmation. Detection of tubulin served as loading control (A) . A549 cells were transfected with either, empty vector (EV) or plasmids encoding JIP4, and infected with PR8 (MOI 5). Cells were lysed every 2 h. Lysates were submitted to Western Blot assay and incubated with antibodies against viral proteins PB1, NS1 and M1, as well as against JIP4 for overexpression confirmation. Detection of tubulin was used as loading control (B) . For analyses of viral mRNA expression, A549 cells were transfected with either siRNAs control or targeting JIP4 and infected with PR8 (MOI 5). Samples were collected every 2 h and progressed for qRT-PCR analyses. Viral mRNAs were normalized over 2 h control group. SPAG9 gene was normalized to MOCK control. Results were statistically analyzed by two-way ANOVA test (C) . **p

    Techniques Used: Expressing, Transfection, Infection, Western Blot, Incubation, Plasmid Preparation, Over Expression, Quantitative RT-PCR

    JIP4 overexpression decreased viral polymerase activity Vero cells were transfected with either, EV or JIP4-expressing plasmids, together with all viral polymerase subunits and a reporter gene. 24 hpt cells were collected for luciferase assay analyses. Activity in EV-transfected cells was arbitrarily set to 100% (A) . A549 cells were transfected with either, siRNAs control or targeting JIP4, and infected with PR8 (MOI 5). Total RNA was collected 4 hpi for strand-specific qRT-PCR (B) . A549 cells were transfected with either, control siRNAs or siRNAs targeting JIP4. After 48 h of transfection, cells were infected with PR8 (MOI 20) and collected for immunofluorescence assay 3 hpi. Localization of JIP4 and NP was analyzed by using specific antibodies, nuclei were stained by DAPI. Percentage of cells with nuclear NP localization was expressed over the total number of cells in the field ( C) . **p
    Figure Legend Snippet: JIP4 overexpression decreased viral polymerase activity Vero cells were transfected with either, EV or JIP4-expressing plasmids, together with all viral polymerase subunits and a reporter gene. 24 hpt cells were collected for luciferase assay analyses. Activity in EV-transfected cells was arbitrarily set to 100% (A) . A549 cells were transfected with either, siRNAs control or targeting JIP4, and infected with PR8 (MOI 5). Total RNA was collected 4 hpi for strand-specific qRT-PCR (B) . A549 cells were transfected with either, control siRNAs or siRNAs targeting JIP4. After 48 h of transfection, cells were infected with PR8 (MOI 20) and collected for immunofluorescence assay 3 hpi. Localization of JIP4 and NP was analyzed by using specific antibodies, nuclei were stained by DAPI. Percentage of cells with nuclear NP localization was expressed over the total number of cells in the field ( C) . **p

    Techniques Used: Over Expression, Activity Assay, Transfection, Expressing, Luciferase, Infection, Quantitative RT-PCR, Immunofluorescence, Staining

    JIP4 presents an anti-viral activity against IAV infection A549 cells were transfected with siRNAs targeting JIP4 or control siRNAs two days prior to infection. Cells were then infected with PR8 IAV (MOI 0.1). Supernatants were collected 8, 24, 36, 48 and 56 hpi and analyzed for viral titers by standard plaque assays. Cells were collected for evaluation of knock-down efficiency via Western blot, tubulin detection served as loading control. Results were statistically analyzed by two-way ANOVA test (A) . A549 cells were transfected with either, empty vector (EV) or plasmids containing JIP4 sequence, and infected with PR8 (MOI 0.1). Supernatants were collected 48 hpi for analysis of viral titers by standard plaque assay. Cells were collected for evaluation of overexpression efficiency by Western blot, tubulin detection served as loading control. Results were statistically analyzed by t-test (B) . A549 cells were transfected with siRNAs targeting JIP4 or control siRNAs and were infected with different IAV strains two days post transfection. PR8, WSN, and H1N1pdm09 were administered with 0.1 MOI. Supernatants were collected 48 hpi for determination of viral replication abilities by standard plaque assays. Results were analyzed by independent t-test per strain (C) . *p
    Figure Legend Snippet: JIP4 presents an anti-viral activity against IAV infection A549 cells were transfected with siRNAs targeting JIP4 or control siRNAs two days prior to infection. Cells were then infected with PR8 IAV (MOI 0.1). Supernatants were collected 8, 24, 36, 48 and 56 hpi and analyzed for viral titers by standard plaque assays. Cells were collected for evaluation of knock-down efficiency via Western blot, tubulin detection served as loading control. Results were statistically analyzed by two-way ANOVA test (A) . A549 cells were transfected with either, empty vector (EV) or plasmids containing JIP4 sequence, and infected with PR8 (MOI 0.1). Supernatants were collected 48 hpi for analysis of viral titers by standard plaque assay. Cells were collected for evaluation of overexpression efficiency by Western blot, tubulin detection served as loading control. Results were statistically analyzed by t-test (B) . A549 cells were transfected with siRNAs targeting JIP4 or control siRNAs and were infected with different IAV strains two days post transfection. PR8, WSN, and H1N1pdm09 were administered with 0.1 MOI. Supernatants were collected 48 hpi for determination of viral replication abilities by standard plaque assays. Results were analyzed by independent t-test per strain (C) . *p

    Techniques Used: Activity Assay, Infection, Transfection, Western Blot, Plasmid Preparation, Sequencing, Plaque Assay, Over Expression

    Phosphorylation of JIP4 S730 results in a decreased viral polymerase activity A549 cells were labeled by using SILAC and infected with low pathogenic IAV strain PR8 (H1N1) or one of two highly pathogenic strains (H5N1 – KAN-1 and H7N7 - FPV). Samples were collected 2, 4, 6 and 8 hpi and analyzed for JIP4 phosphorylation. Relative phosphorylation of JIP4 at S730 at different time points compared to non-infected cells is depicted after normalization to respective JIP4 protein amounts (A) . Vero cells were transfected with plasmids expressing either, EV, WT JIP4, or JIP4 phospho-mutants S730A or S730E, together with all viral polymerase subunits and a reporter gene. 24 hpt, cells were collected for luciferase assay analysis. Activity in EV-transfected cells was arbitrarily set to 100% and all groups were statistically compared with EV-transfected cells by one-way ANOVA test. Overexpression was confirmed by Western blot (B) . Vero cells were transfected with either, EV or plasmids expressing WT JIP4, together with all viral polymerase subunits and a reporter gene. After 4 h of transfection, medium was replaced by fresh medium supplemented with vehicle or 10 μM MEK inhibitor U0126. Luciferase assay was performed 24 hpt. Activity in EV-transfected cells group was arbitrarily set to 100%. All groups were compared by two-way ANOVA test. (C) . ***p
    Figure Legend Snippet: Phosphorylation of JIP4 S730 results in a decreased viral polymerase activity A549 cells were labeled by using SILAC and infected with low pathogenic IAV strain PR8 (H1N1) or one of two highly pathogenic strains (H5N1 – KAN-1 and H7N7 - FPV). Samples were collected 2, 4, 6 and 8 hpi and analyzed for JIP4 phosphorylation. Relative phosphorylation of JIP4 at S730 at different time points compared to non-infected cells is depicted after normalization to respective JIP4 protein amounts (A) . Vero cells were transfected with plasmids expressing either, EV, WT JIP4, or JIP4 phospho-mutants S730A or S730E, together with all viral polymerase subunits and a reporter gene. 24 hpt, cells were collected for luciferase assay analysis. Activity in EV-transfected cells was arbitrarily set to 100% and all groups were statistically compared with EV-transfected cells by one-way ANOVA test. Overexpression was confirmed by Western blot (B) . Vero cells were transfected with either, EV or plasmids expressing WT JIP4, together with all viral polymerase subunits and a reporter gene. After 4 h of transfection, medium was replaced by fresh medium supplemented with vehicle or 10 μM MEK inhibitor U0126. Luciferase assay was performed 24 hpt. Activity in EV-transfected cells group was arbitrarily set to 100%. All groups were compared by two-way ANOVA test. (C) . ***p

    Techniques Used: Activity Assay, Labeling, Infection, Transfection, Expressing, Luciferase, Over Expression, Western Blot

    The anti-viral role of JIP4 does not correlate with changes in IFN responses or JNK/p38 activation A549 cells were transfected with either, control siRNAs or siRNAs targeting JIP4, and infected with PR8 (MOI 5). Cells were collected every two hours for qRT-PCR analysis focusing on IFN and ISG mRNA expression. Results were statistically analyzed by two-way ANOVA test (A) . A549 cells were transfected with either, siRNAs control or targeting JIP4, and stimulated with IFNß for 2 or 4 h. ISG mRNA levels were analyzed by qRT-PCR. Results were statistically analyzed by two-way ANOVA test (B) . Results are depicted as n-fold expression over MOCK/vehicle after normalization to GAPDH expression (A, B) . A549 cells were transfected with either, siRNAs control or targeting JIP4, and infected with VSV (MOI 0.01). Supernatants were collected 24 hpi and virus replication was analyzed by standard plaque assays. Results are expressed in PFU/ml and statistically analyzed by t-test (C) . For analysis of JNK and p38 activity, A549 cells were transfected with either, EV or JIP4-expressing plasmids, and incubated for 24 h. Cells were then further transfected with c/vRNAs to stimulate activation of JNK and p38 kinases. After 3 h of stimulation, cells were lysed for Western blot analysis and activation of MAPKs was determined by using pJNK and p-p38 antibodies. Detection of p38, JNK and tubulin served as loading controls, successful overexpression was confirmed by JIP4 detection (D) . All Western Blot images are representative of three independent experiments. Graphs are a compilation of two independent experiments.
    Figure Legend Snippet: The anti-viral role of JIP4 does not correlate with changes in IFN responses or JNK/p38 activation A549 cells were transfected with either, control siRNAs or siRNAs targeting JIP4, and infected with PR8 (MOI 5). Cells were collected every two hours for qRT-PCR analysis focusing on IFN and ISG mRNA expression. Results were statistically analyzed by two-way ANOVA test (A) . A549 cells were transfected with either, siRNAs control or targeting JIP4, and stimulated with IFNß for 2 or 4 h. ISG mRNA levels were analyzed by qRT-PCR. Results were statistically analyzed by two-way ANOVA test (B) . Results are depicted as n-fold expression over MOCK/vehicle after normalization to GAPDH expression (A, B) . A549 cells were transfected with either, siRNAs control or targeting JIP4, and infected with VSV (MOI 0.01). Supernatants were collected 24 hpi and virus replication was analyzed by standard plaque assays. Results are expressed in PFU/ml and statistically analyzed by t-test (C) . For analysis of JNK and p38 activity, A549 cells were transfected with either, EV or JIP4-expressing plasmids, and incubated for 24 h. Cells were then further transfected with c/vRNAs to stimulate activation of JNK and p38 kinases. After 3 h of stimulation, cells were lysed for Western blot analysis and activation of MAPKs was determined by using pJNK and p-p38 antibodies. Detection of p38, JNK and tubulin served as loading controls, successful overexpression was confirmed by JIP4 detection (D) . All Western Blot images are representative of three independent experiments. Graphs are a compilation of two independent experiments.

    Techniques Used: Activation Assay, Transfection, Infection, Quantitative RT-PCR, Expressing, Activity Assay, Incubation, Western Blot, Over Expression

    27) Product Images from "Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti"

    Article Title: Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2019.00266

    LGR1 knockdown efficiency in adult A. aegypti . Second instar larvae were fed with Escherichia coli expressing LGR1 dsRNA or control dsRNA (empty L4440 vector). After feeding, LGR1 transcript knockdown efficiency was determined in panel (A) whole 4th-instar larvae and one-day old adults by quantitative RT-PCR. LGR1 immunoreactivity was examined in control and LGR1 knockdown treatments within (B–F) the testes of virgin one-day adult males (LGR1, green; nuclei, blue). (A) LGR1 transcript levels of 4th instar larvae and newly emerged adults fed with LGR1 dsRNA were significantly reduced compared to control dsRNA-fed mosquitoes. (B) Quantification of LGR1 immunoreactivity from control and dsLGR1-treated larvae confirms downregulation. Representative confocal images demonstrate reduced LGR1 immunofluorescence in cysts (dotted outline) containing late-staged spermatids (Lst) when comparing (C,E) control and (D,F) dsLGR1-treated mosquitoes. All data are presented as mean ± SEM ( n = 3 in panel A , n = 11–13 in panel B ) with asterisks representing significant differences between control and dsLGR1-treated mosquitoes as determined using an unpaired t -test: ∗ P
    Figure Legend Snippet: LGR1 knockdown efficiency in adult A. aegypti . Second instar larvae were fed with Escherichia coli expressing LGR1 dsRNA or control dsRNA (empty L4440 vector). After feeding, LGR1 transcript knockdown efficiency was determined in panel (A) whole 4th-instar larvae and one-day old adults by quantitative RT-PCR. LGR1 immunoreactivity was examined in control and LGR1 knockdown treatments within (B–F) the testes of virgin one-day adult males (LGR1, green; nuclei, blue). (A) LGR1 transcript levels of 4th instar larvae and newly emerged adults fed with LGR1 dsRNA were significantly reduced compared to control dsRNA-fed mosquitoes. (B) Quantification of LGR1 immunoreactivity from control and dsLGR1-treated larvae confirms downregulation. Representative confocal images demonstrate reduced LGR1 immunofluorescence in cysts (dotted outline) containing late-staged spermatids (Lst) when comparing (C,E) control and (D,F) dsLGR1-treated mosquitoes. All data are presented as mean ± SEM ( n = 3 in panel A , n = 11–13 in panel B ) with asterisks representing significant differences between control and dsLGR1-treated mosquitoes as determined using an unpaired t -test: ∗ P

    Techniques Used: Expressing, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence

    Effects of LGR1 knockdown on spermatozoa yield, flagellar length and reproductive success of adult male A. aegypti . Second instar larvae were fed with E. coli expressing LGR1 dsRNA or control dsRNA (empty L4440 vector) and the effects of knockdown on sperm yield, flagellar length and male fertility were analyzed in adult stages. Relative to controls, (A–C) dsLGR1-fed mosquitoes had significantly less spermatozoa in testes and in the overall reproductive tract (testes + seminal vesicle), while the number of spermatozoa in the seminal vesicle showed a similar trend but was not significant. (D) Flagellar lengths from spermatozoa of dsLGR1-treated mosquitoes were significantly shorter than flagella from control mosquitoes. Representative images showing flagellar lengths (arrowheads indicate beginning and end of flagellum) of spermatozoa collected from (E) control- and (F) dsLGR1-treated mosquitoes (nuclei, blue). (G,H) No differences in egg yield were observed between females mated with control or dsLGRl-treated males; however, the percentage of larvae hatched from eggs was significantly reduced in matings that involved male LGR1 knockdown treatments. All data are presented as mean ± SEM ( n = 17 in panel A–C , n = 14–15 in panel D , n = 13 in panel G,H ) with asterisks representing significant differences between control and dsLGR1-treated mosquitoes as determined using an unpaired t -test: ∗∗ P
    Figure Legend Snippet: Effects of LGR1 knockdown on spermatozoa yield, flagellar length and reproductive success of adult male A. aegypti . Second instar larvae were fed with E. coli expressing LGR1 dsRNA or control dsRNA (empty L4440 vector) and the effects of knockdown on sperm yield, flagellar length and male fertility were analyzed in adult stages. Relative to controls, (A–C) dsLGR1-fed mosquitoes had significantly less spermatozoa in testes and in the overall reproductive tract (testes + seminal vesicle), while the number of spermatozoa in the seminal vesicle showed a similar trend but was not significant. (D) Flagellar lengths from spermatozoa of dsLGR1-treated mosquitoes were significantly shorter than flagella from control mosquitoes. Representative images showing flagellar lengths (arrowheads indicate beginning and end of flagellum) of spermatozoa collected from (E) control- and (F) dsLGR1-treated mosquitoes (nuclei, blue). (G,H) No differences in egg yield were observed between females mated with control or dsLGRl-treated males; however, the percentage of larvae hatched from eggs was significantly reduced in matings that involved male LGR1 knockdown treatments. All data are presented as mean ± SEM ( n = 17 in panel A–C , n = 14–15 in panel D , n = 13 in panel G,H ) with asterisks representing significant differences between control and dsLGR1-treated mosquitoes as determined using an unpaired t -test: ∗∗ P

    Techniques Used: Expressing, Plasmid Preparation

    28) Product Images from "Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti"

    Article Title: Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2019.00266

    LGR1 knockdown efficiency in adult A. aegypti . Second instar larvae were fed with Escherichia coli expressing LGR1 dsRNA or control dsRNA (empty L4440 vector). After feeding, LGR1 transcript knockdown efficiency was determined in panel (A) whole 4th-instar larvae and one-day old adults by quantitative RT-PCR. LGR1 immunoreactivity was examined in control and LGR1 knockdown treatments within (B–F) the testes of virgin one-day adult males (LGR1, green; nuclei, blue). (A) LGR1 transcript levels of 4th instar larvae and newly emerged adults fed with LGR1 dsRNA were significantly reduced compared to control dsRNA-fed mosquitoes. (B) Quantification of LGR1 immunoreactivity from control and dsLGR1-treated larvae confirms downregulation. Representative confocal images demonstrate reduced LGR1 immunofluorescence in cysts (dotted outline) containing late-staged spermatids (Lst) when comparing (C,E) control and (D,F) dsLGR1-treated mosquitoes. All data are presented as mean ± SEM ( n = 3 in panel A , n = 11–13 in panel B ) with asterisks representing significant differences between control and dsLGR1-treated mosquitoes as determined using an unpaired t -test: ∗ P
    Figure Legend Snippet: LGR1 knockdown efficiency in adult A. aegypti . Second instar larvae were fed with Escherichia coli expressing LGR1 dsRNA or control dsRNA (empty L4440 vector). After feeding, LGR1 transcript knockdown efficiency was determined in panel (A) whole 4th-instar larvae and one-day old adults by quantitative RT-PCR. LGR1 immunoreactivity was examined in control and LGR1 knockdown treatments within (B–F) the testes of virgin one-day adult males (LGR1, green; nuclei, blue). (A) LGR1 transcript levels of 4th instar larvae and newly emerged adults fed with LGR1 dsRNA were significantly reduced compared to control dsRNA-fed mosquitoes. (B) Quantification of LGR1 immunoreactivity from control and dsLGR1-treated larvae confirms downregulation. Representative confocal images demonstrate reduced LGR1 immunofluorescence in cysts (dotted outline) containing late-staged spermatids (Lst) when comparing (C,E) control and (D,F) dsLGR1-treated mosquitoes. All data are presented as mean ± SEM ( n = 3 in panel A , n = 11–13 in panel B ) with asterisks representing significant differences between control and dsLGR1-treated mosquitoes as determined using an unpaired t -test: ∗ P

    Techniques Used: Expressing, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence

    Effects of LGR1 knockdown on spermatozoa yield, flagellar length and reproductive success of adult male A. aegypti . Second instar larvae were fed with E. coli expressing LGR1 dsRNA or control dsRNA (empty L4440 vector) and the effects of knockdown on sperm yield, flagellar length and male fertility were analyzed in adult stages. Relative to controls, (A–C) dsLGR1-fed mosquitoes had significantly less spermatozoa in testes and in the overall reproductive tract (testes + seminal vesicle), while the number of spermatozoa in the seminal vesicle showed a similar trend but was not significant. (D) Flagellar lengths from spermatozoa of dsLGR1-treated mosquitoes were significantly shorter than flagella from control mosquitoes. Representative images showing flagellar lengths (arrowheads indicate beginning and end of flagellum) of spermatozoa collected from (E) control- and (F) dsLGR1-treated mosquitoes (nuclei, blue). (G,H) No differences in egg yield were observed between females mated with control or dsLGRl-treated males; however, the percentage of larvae hatched from eggs was significantly reduced in matings that involved male LGR1 knockdown treatments. All data are presented as mean ± SEM ( n = 17 in panel A–C , n = 14–15 in panel D , n = 13 in panel G,H ) with asterisks representing significant differences between control and dsLGR1-treated mosquitoes as determined using an unpaired t -test: ∗∗ P
    Figure Legend Snippet: Effects of LGR1 knockdown on spermatozoa yield, flagellar length and reproductive success of adult male A. aegypti . Second instar larvae were fed with E. coli expressing LGR1 dsRNA or control dsRNA (empty L4440 vector) and the effects of knockdown on sperm yield, flagellar length and male fertility were analyzed in adult stages. Relative to controls, (A–C) dsLGR1-fed mosquitoes had significantly less spermatozoa in testes and in the overall reproductive tract (testes + seminal vesicle), while the number of spermatozoa in the seminal vesicle showed a similar trend but was not significant. (D) Flagellar lengths from spermatozoa of dsLGR1-treated mosquitoes were significantly shorter than flagella from control mosquitoes. Representative images showing flagellar lengths (arrowheads indicate beginning and end of flagellum) of spermatozoa collected from (E) control- and (F) dsLGR1-treated mosquitoes (nuclei, blue). (G,H) No differences in egg yield were observed between females mated with control or dsLGRl-treated males; however, the percentage of larvae hatched from eggs was significantly reduced in matings that involved male LGR1 knockdown treatments. All data are presented as mean ± SEM ( n = 17 in panel A–C , n = 14–15 in panel D , n = 13 in panel G,H ) with asterisks representing significant differences between control and dsLGR1-treated mosquitoes as determined using an unpaired t -test: ∗∗ P

    Techniques Used: Expressing, Plasmid Preparation

    29) Product Images from "Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies"

    Article Title: Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies

    Journal: Genes

    doi: 10.3390/genes10020079

    Functional annotation of the Tni-FNL ( Trichoplusia ni ), B. mori and D. melanogaster results comparison. The circos plot describes the shared cellular components, molecular functions and biological processes among the three species.
    Figure Legend Snippet: Functional annotation of the Tni-FNL ( Trichoplusia ni ), B. mori and D. melanogaster results comparison. The circos plot describes the shared cellular components, molecular functions and biological processes among the three species.

    Techniques Used: Functional Assay

    30) Product Images from "Towards deorphanizing G protein-coupled receptors of Schistosoma mansoni using the MALAR yeast two-hybrid system"

    Article Title: Towards deorphanizing G protein-coupled receptors of Schistosoma mansoni using the MALAR yeast two-hybrid system

    Journal: Parasitology

    doi: 10.1017/S0031182019001756

    Full-length expression of S. mansoni GPCRs in S. cerevisiae . Wild type or optimized CDSs of GPCRs were transformed into yeast strain AH109. RNA was isolated and transcribed into cDNA. Specific primer pairs were used to amplify corresponding GPCRs (numbers 1-7) by RT-PCR, and resulting amplicons were size-separated on a 1% agarose gel (M = marker, bp = base pair). The absence of reverse transcriptase (-RT) during cDNA synthesis or cDNA synthesis without template (H2O) served as negative controls. RNA isolated from S. mansoni served as a positive control for full-length transcripts. In each case, amplicons of the expected sizes were obtained.
    Figure Legend Snippet: Full-length expression of S. mansoni GPCRs in S. cerevisiae . Wild type or optimized CDSs of GPCRs were transformed into yeast strain AH109. RNA was isolated and transcribed into cDNA. Specific primer pairs were used to amplify corresponding GPCRs (numbers 1-7) by RT-PCR, and resulting amplicons were size-separated on a 1% agarose gel (M = marker, bp = base pair). The absence of reverse transcriptase (-RT) during cDNA synthesis or cDNA synthesis without template (H2O) served as negative controls. RNA isolated from S. mansoni served as a positive control for full-length transcripts. In each case, amplicons of the expected sizes were obtained.

    Techniques Used: Expressing, Transformation Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Positive Control

    31) Product Images from "Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump"

    Article Title: Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0243082

    Time-kill curves of resveratrol and chlorhexidine against A . baumannii . Time-kill curves of resveratrol 128 mg/L in combination with 0.25x MIC of chlorhexidine (4 or 8 mg/L) against A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21(d). Mean values of viable cells were plotted with error bars representing standard deviations. All experiments were performed in triplicate and the detection limit of the viable cells is 10 2 CFU/mL.
    Figure Legend Snippet: Time-kill curves of resveratrol and chlorhexidine against A . baumannii . Time-kill curves of resveratrol 128 mg/L in combination with 0.25x MIC of chlorhexidine (4 or 8 mg/L) against A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21(d). Mean values of viable cells were plotted with error bars representing standard deviations. All experiments were performed in triplicate and the detection limit of the viable cells is 10 2 CFU/mL.

    Techniques Used:

    Effect of resveratrol in the presence of chlorhexidine on ethidium bromide accumulation in A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21 (d). The fluorescence of ethidium bromide was measured in the presence of glucose with (filled triangles) or without (open circles) chlorhexidine (8 mg/L) and after addition of CCCP (open squares) or resveratrol (open triangles). Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.
    Figure Legend Snippet: Effect of resveratrol in the presence of chlorhexidine on ethidium bromide accumulation in A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21 (d). The fluorescence of ethidium bromide was measured in the presence of glucose with (filled triangles) or without (open circles) chlorhexidine (8 mg/L) and after addition of CCCP (open squares) or resveratrol (open triangles). Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.

    Techniques Used: Fluorescence

    Effect of chlorhexidine and resveratrol alone or in combination on expression of efflux pump and efflux pump regulator genes in A . baumannii strain L14. RT-qPCR assay of adeB (a), adeR (b), adeS (c), adeJ (d), adeG (e), abeS (f), and aceI (g) expression in the presence of MHB control, DMSO control, either chlorhexidine (8 mg/L) or resveratrol (128 mg/L) and in the combination of chlorhexidine with resveratrol. Relative number of transcripts of each gene was normalized to 16S rRNA expression in each condition and calculated using the 2 -ΔΔct method compared to the expression level in MHB control. The relative number of transcripts of each gene was plotted with error bars representing standard deviations. All experiments were performed in triplicate. p- values were calculated using ANOVA (#, p -value
    Figure Legend Snippet: Effect of chlorhexidine and resveratrol alone or in combination on expression of efflux pump and efflux pump regulator genes in A . baumannii strain L14. RT-qPCR assay of adeB (a), adeR (b), adeS (c), adeJ (d), adeG (e), abeS (f), and aceI (g) expression in the presence of MHB control, DMSO control, either chlorhexidine (8 mg/L) or resveratrol (128 mg/L) and in the combination of chlorhexidine with resveratrol. Relative number of transcripts of each gene was normalized to 16S rRNA expression in each condition and calculated using the 2 -ΔΔct method compared to the expression level in MHB control. The relative number of transcripts of each gene was plotted with error bars representing standard deviations. All experiments were performed in triplicate. p- values were calculated using ANOVA (#, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of resveratrol on ethidium bromide accumulation in A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21 (d). The fluorescence of ethidium bromide was measured in the presence of glucose and after addition of CCCP (filled squares), resveratrol (filled triangles), or control with no addition of any proton coupler agent (fill circles). Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.
    Figure Legend Snippet: Effect of resveratrol on ethidium bromide accumulation in A . baumannii strain AC152 (a), AC154 (b), L14 (c), and L21 (d). The fluorescence of ethidium bromide was measured in the presence of glucose and after addition of CCCP (filled squares), resveratrol (filled triangles), or control with no addition of any proton coupler agent (fill circles). Relative fluorescence units were plotted with error bars representing standard deviations. All experiments were performed in triplicate.

    Techniques Used: Fluorescence

    32) Product Images from "ETV7 represses a subset of interferon-stimulated genes that restrict influenza viruses"

    Article Title: ETV7 represses a subset of interferon-stimulated genes that restrict influenza viruses

    Journal: bioRxiv

    doi: 10.1101/851543

    ETV7 loss enhances expression of specific ISGs. A) ISG15 and ETV7 mRNA levels in A549 lung epithelial cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). B) Percentage of A549 cells expressing GFP from IFNrsp reporter after knockout of ETV7 and IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=3, statistical analysis represents p-values for both of the two ETV7 KO sgRNAs compared to a non-targeting control). C) mRNA levels of ETV7 in non-targeting control and ETV7 KO A549 cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). D,E) Representative genes were chosen from the groups D) most affected by ETV7 (Group I) and E) least affected (Groups II and III) in the RNA sequencing analysis ( Fig. 4B ). Each gene’s potential ISRE sequences (ETS sites highlighted in yellow) are shown, along with its mRNA levels in control and ETV7 KO cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). For all panels: Data shown are representative of two independent experiments. P-values calculated using unpaired, two-tailed Student’s t-tests (*p
    Figure Legend Snippet: ETV7 loss enhances expression of specific ISGs. A) ISG15 and ETV7 mRNA levels in A549 lung epithelial cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). B) Percentage of A549 cells expressing GFP from IFNrsp reporter after knockout of ETV7 and IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=3, statistical analysis represents p-values for both of the two ETV7 KO sgRNAs compared to a non-targeting control). C) mRNA levels of ETV7 in non-targeting control and ETV7 KO A549 cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). D,E) Representative genes were chosen from the groups D) most affected by ETV7 (Group I) and E) least affected (Groups II and III) in the RNA sequencing analysis ( Fig. 4B ). Each gene’s potential ISRE sequences (ETS sites highlighted in yellow) are shown, along with its mRNA levels in control and ETV7 KO cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). For all panels: Data shown are representative of two independent experiments. P-values calculated using unpaired, two-tailed Student’s t-tests (*p

    Techniques Used: Expressing, Knock-Out, RNA Sequencing Assay, Two Tailed Test

    33) Product Images from "Slaying the last unicorn - discovery of histones in the microalga Nanochlorum eucaryotum"

    Article Title: Slaying the last unicorn - discovery of histones in the microalga Nanochlorum eucaryotum

    Journal: bioRxiv

    doi: 10.1101/2020.10.22.350256

    Abundance of histone transcripts compared to the remainder of de novo-assembled transcripts in Nanochlorum eucaryotum (SAG 55.87) and five other eukaryotes, including three algae (C. reinhardtii, D. tertiolecta, C. zofingiensis). Abundance of the most highly expressed histone transcript (solid lines) and the summed abundance of all histone transcripts (dashed lines) is indicated for each species. See Methods for details on the underlying RNA-Seq data, assembly and quantification process.
    Figure Legend Snippet: Abundance of histone transcripts compared to the remainder of de novo-assembled transcripts in Nanochlorum eucaryotum (SAG 55.87) and five other eukaryotes, including three algae (C. reinhardtii, D. tertiolecta, C. zofingiensis). Abundance of the most highly expressed histone transcript (solid lines) and the summed abundance of all histone transcripts (dashed lines) is indicated for each species. See Methods for details on the underlying RNA-Seq data, assembly and quantification process.

    Techniques Used: RNA Sequencing Assay

    34) Product Images from "DNA mismatch repair controls the host innate response and cell fate after influenza virus infection"

    Article Title: DNA mismatch repair controls the host innate response and cell fate after influenza virus infection

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0509-3

    Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.
    Figure Legend Snippet: Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.

    Techniques Used: Activity Assay, Expressing, Western Blot, Preserving, Infection, Transfection, Two Tailed Test

    35) Product Images from "DNA mismatch repair controls the host innate response and cell fate after influenza virus infection"

    Article Title: DNA mismatch repair controls the host innate response and cell fate after influenza virus infection

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0509-3

    Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.
    Figure Legend Snippet: Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.

    Techniques Used: Activity Assay, Expressing, Western Blot, Preserving, Infection, Transfection, Two Tailed Test

    36) Product Images from "Haemophilus influenzae persists in biofilm communities in a smoke-exposed ferret model of COPD"

    Article Title: Haemophilus influenzae persists in biofilm communities in a smoke-exposed ferret model of COPD

    Journal: ERJ Open Research

    doi: 10.1183/23120541.00200-2020

    a) Confocal immunofluorescent imaging of ferret lungs. Smoke exposure enables non-typeable Haemophilus influenzae (NTHi) to form aggregates within the lungs. Nuclei were stained with DAPI (blue) and bacteria were stained with anti-NTHi polyclonal antibodies conjugated to Alexa 488 (green). Infected smoke-exposed animals exhibit bacterial aggregates both in the airway and lung parenchyma. By contrast, infected air control animals displayed few punctate NTHi in the lungs. Both uninfected smoke and air controls displayed no NTHi. Scale bars=50 µm. b) Confocal imaging of sialic acid lectin staining of NTHi biofilms. Nuclei were stained with DAPI (blue), bacteria were stained with anti-NTHi polyclonal antibodies conjugated to Alexa 488 (green), sialic α(2,3) galactose was stained with Maackia amurensis lectin (MAA) conjugated to Texas red (red), and Neu5Acα(2,6) Gal/GalNAc was stained with Sambucus nigra lectin (SNA) conjugated to Texas red (red). Images were taken at 20× zoom. Sialic acid staining overlaps with regions stained for NTHi suggesting biofilm formation is occurring within the airways during exacerbation; this is indicated by the yellow in the composite images. c) Relative expression of NTHi biofilm genes determined by reverse transcriptase quantitative PCR (RT-qPCR) in infected smoke-exposed animals. Three genes associated with NTHi biofilm growth were all significantly upregulated in comparison to the housekeeping gene omp26. Of interest, dps , a DNA-binding protein associated with resistance to environmental stress was very highly upregulated. hktE, the gene encoding for NTHi catalase, was also very highly upregulated, giving indications that NTHi was growing in a highly oxidatively stressful environment in the COPD lung. All samples were run in triplicate. Mean± sem . n=4. Scale bars: 50 μm.
    Figure Legend Snippet: a) Confocal immunofluorescent imaging of ferret lungs. Smoke exposure enables non-typeable Haemophilus influenzae (NTHi) to form aggregates within the lungs. Nuclei were stained with DAPI (blue) and bacteria were stained with anti-NTHi polyclonal antibodies conjugated to Alexa 488 (green). Infected smoke-exposed animals exhibit bacterial aggregates both in the airway and lung parenchyma. By contrast, infected air control animals displayed few punctate NTHi in the lungs. Both uninfected smoke and air controls displayed no NTHi. Scale bars=50 µm. b) Confocal imaging of sialic acid lectin staining of NTHi biofilms. Nuclei were stained with DAPI (blue), bacteria were stained with anti-NTHi polyclonal antibodies conjugated to Alexa 488 (green), sialic α(2,3) galactose was stained with Maackia amurensis lectin (MAA) conjugated to Texas red (red), and Neu5Acα(2,6) Gal/GalNAc was stained with Sambucus nigra lectin (SNA) conjugated to Texas red (red). Images were taken at 20× zoom. Sialic acid staining overlaps with regions stained for NTHi suggesting biofilm formation is occurring within the airways during exacerbation; this is indicated by the yellow in the composite images. c) Relative expression of NTHi biofilm genes determined by reverse transcriptase quantitative PCR (RT-qPCR) in infected smoke-exposed animals. Three genes associated with NTHi biofilm growth were all significantly upregulated in comparison to the housekeeping gene omp26. Of interest, dps , a DNA-binding protein associated with resistance to environmental stress was very highly upregulated. hktE, the gene encoding for NTHi catalase, was also very highly upregulated, giving indications that NTHi was growing in a highly oxidatively stressful environment in the COPD lung. All samples were run in triplicate. Mean± sem . n=4. Scale bars: 50 μm.

    Techniques Used: Imaging, Staining, Infection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Binding Assay

    37) Product Images from "Haemophilus influenzae persists in biofilm communities in a smoke-exposed ferret model of COPD"

    Article Title: Haemophilus influenzae persists in biofilm communities in a smoke-exposed ferret model of COPD

    Journal: ERJ Open Research

    doi: 10.1183/23120541.00200-2020

    a) Confocal immunofluorescent imaging of ferret lungs. Smoke exposure enables non-typeable Haemophilus influenzae (NTHi) to form aggregates within the lungs. Nuclei were stained with DAPI (blue) and bacteria were stained with anti-NTHi polyclonal antibodies conjugated to Alexa 488 (green). Infected smoke-exposed animals exhibit bacterial aggregates both in the airway and lung parenchyma. By contrast, infected air control animals displayed few punctate NTHi in the lungs. Both uninfected smoke and air controls displayed no NTHi. Scale bars=50 µm. b) Confocal imaging of sialic acid lectin staining of NTHi biofilms. Nuclei were stained with DAPI (blue), bacteria were stained with anti-NTHi polyclonal antibodies conjugated to Alexa 488 (green), sialic α(2,3) galactose was stained with Maackia amurensis lectin (MAA) conjugated to Texas red (red), and Neu5Acα(2,6) Gal/GalNAc was stained with Sambucus nigra lectin (SNA) conjugated to Texas red (red). Images were taken at 20× zoom. Sialic acid staining overlaps with regions stained for NTHi suggesting biofilm formation is occurring within the airways during exacerbation; this is indicated by the yellow in the composite images. c) Relative expression of NTHi biofilm genes determined by reverse transcriptase quantitative PCR (RT-qPCR) in infected smoke-exposed animals. Three genes associated with NTHi biofilm growth were all significantly upregulated in comparison to the housekeeping gene omp26. Of interest, dps , a DNA-binding protein associated with resistance to environmental stress was very highly upregulated. hktE, the gene encoding for NTHi catalase, was also very highly upregulated, giving indications that NTHi was growing in a highly oxidatively stressful environment in the COPD lung. All samples were run in triplicate. Mean± sem . n=4. Scale bars: 50 μm.
    Figure Legend Snippet: a) Confocal immunofluorescent imaging of ferret lungs. Smoke exposure enables non-typeable Haemophilus influenzae (NTHi) to form aggregates within the lungs. Nuclei were stained with DAPI (blue) and bacteria were stained with anti-NTHi polyclonal antibodies conjugated to Alexa 488 (green). Infected smoke-exposed animals exhibit bacterial aggregates both in the airway and lung parenchyma. By contrast, infected air control animals displayed few punctate NTHi in the lungs. Both uninfected smoke and air controls displayed no NTHi. Scale bars=50 µm. b) Confocal imaging of sialic acid lectin staining of NTHi biofilms. Nuclei were stained with DAPI (blue), bacteria were stained with anti-NTHi polyclonal antibodies conjugated to Alexa 488 (green), sialic α(2,3) galactose was stained with Maackia amurensis lectin (MAA) conjugated to Texas red (red), and Neu5Acα(2,6) Gal/GalNAc was stained with Sambucus nigra lectin (SNA) conjugated to Texas red (red). Images were taken at 20× zoom. Sialic acid staining overlaps with regions stained for NTHi suggesting biofilm formation is occurring within the airways during exacerbation; this is indicated by the yellow in the composite images. c) Relative expression of NTHi biofilm genes determined by reverse transcriptase quantitative PCR (RT-qPCR) in infected smoke-exposed animals. Three genes associated with NTHi biofilm growth were all significantly upregulated in comparison to the housekeeping gene omp26. Of interest, dps , a DNA-binding protein associated with resistance to environmental stress was very highly upregulated. hktE, the gene encoding for NTHi catalase, was also very highly upregulated, giving indications that NTHi was growing in a highly oxidatively stressful environment in the COPD lung. All samples were run in triplicate. Mean± sem . n=4. Scale bars: 50 μm.

    Techniques Used: Imaging, Staining, Infection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Binding Assay

    38) Product Images from "DNA mismatch repair controls the host innate response and cell fate after influenza virus infection"

    Article Title: DNA mismatch repair controls the host innate response and cell fate after influenza virus infection

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0509-3

    Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.
    Figure Legend Snippet: Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.

    Techniques Used: Activity Assay, Expressing, Western Blot, Preserving, Infection, Transfection, Two Tailed Test

    39) Product Images from "ETV7 represses a subset of interferon-stimulated genes that restrict influenza viruses"

    Article Title: ETV7 represses a subset of interferon-stimulated genes that restrict influenza viruses

    Journal: bioRxiv

    doi: 10.1101/851543

    ETV7 differentially regulates ISGs during the type I IFN response based on ISRE-related regulatory elements. A) Dendrogram of genes most differentially expressed in cells overexpressing either a control protein (mCherry) or ETV7 before and after IFN-α treatment (100 U/mL, 9 h) as measured using RNA sequencing. Three independent, biological replicates per condition. Red box highlights control samples, yellow box highlights ETV7-expressing samples, shading indicates IFN-stimulated samples. The box width indicates the linkage distance between samples before and after IFN, indicating control cells’ transcriptional profile is more diverged after IFN treatment compared to ETV7-expressing cells. B) Heat map displaying RNA levels of genes upregulated at least two-fold following IFN-α treatment (100 U/mL, 9 h) in control cells. Expression was normalized to control cells after IFN treatment (averaged across replicates). Yellow = downregulated, blue = upregulated. C,D) Motif counts in promoter regions (−1000 bp, +500 bp) for the genes most and least affected by ETV7 overexpression in the RNA sequencing results. ETS sites (GGAA) highlighted in yellow. P-values calculated using unpaired, two-tailed Mann-Whitney U tests (*p
    Figure Legend Snippet: ETV7 differentially regulates ISGs during the type I IFN response based on ISRE-related regulatory elements. A) Dendrogram of genes most differentially expressed in cells overexpressing either a control protein (mCherry) or ETV7 before and after IFN-α treatment (100 U/mL, 9 h) as measured using RNA sequencing. Three independent, biological replicates per condition. Red box highlights control samples, yellow box highlights ETV7-expressing samples, shading indicates IFN-stimulated samples. The box width indicates the linkage distance between samples before and after IFN, indicating control cells’ transcriptional profile is more diverged after IFN treatment compared to ETV7-expressing cells. B) Heat map displaying RNA levels of genes upregulated at least two-fold following IFN-α treatment (100 U/mL, 9 h) in control cells. Expression was normalized to control cells after IFN treatment (averaged across replicates). Yellow = downregulated, blue = upregulated. C,D) Motif counts in promoter regions (−1000 bp, +500 bp) for the genes most and least affected by ETV7 overexpression in the RNA sequencing results. ETS sites (GGAA) highlighted in yellow. P-values calculated using unpaired, two-tailed Mann-Whitney U tests (*p

    Techniques Used: RNA Sequencing Assay, Expressing, Over Expression, Two Tailed Test, MANN-WHITNEY

    ETV7 loss enhances expression of specific ISGs. A) ISG15 and ETV7 mRNA levels in A549 lung epithelial cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). B) Percentage of A549 cells expressing GFP from IFNrsp reporter after knockout of ETV7 and IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=3, statistical analysis represents p-values for both of the two ETV7 KO sgRNAs compared to a non-targeting control). C) mRNA levels of ETV7 in non-targeting control and ETV7 KO A549 cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). D,E) Representative genes were chosen from the groups D) most affected by ETV7 (Group I) and E) least affected (Groups II and III) in the RNA sequencing analysis ( Fig. 4B ). Each gene’s potential ISRE sequences (ETS sites highlighted in yellow) are shown, along with its mRNA levels in control and ETV7 KO cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). For all panels: Data shown are representative of two independent experiments. P-values calculated using unpaired, two-tailed Student’s t-tests (*p
    Figure Legend Snippet: ETV7 loss enhances expression of specific ISGs. A) ISG15 and ETV7 mRNA levels in A549 lung epithelial cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). B) Percentage of A549 cells expressing GFP from IFNrsp reporter after knockout of ETV7 and IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=3, statistical analysis represents p-values for both of the two ETV7 KO sgRNAs compared to a non-targeting control). C) mRNA levels of ETV7 in non-targeting control and ETV7 KO A549 cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). D,E) Representative genes were chosen from the groups D) most affected by ETV7 (Group I) and E) least affected (Groups II and III) in the RNA sequencing analysis ( Fig. 4B ). Each gene’s potential ISRE sequences (ETS sites highlighted in yellow) are shown, along with its mRNA levels in control and ETV7 KO cells after IFN-α treatment (1000 U/mL, 6 h) (data shown as mean ± SD, n=4). For all panels: Data shown are representative of two independent experiments. P-values calculated using unpaired, two-tailed Student’s t-tests (*p

    Techniques Used: Expressing, Knock-Out, RNA Sequencing Assay, Two Tailed Test

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    RNA Sequencing Assay:

    Article Title: Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting
    Article Snippet: .. For RNA-seq of C1R-CD1d parental cells, the same procedures were followed, except for the fact that libraries were prepared .using the NEBNext poly(A) mRNA Magnetic Isolation Module and the NEBNext Ultra RNA Library Prep kit for Illumina (New Engand Biolabs) and sequenced on an Illumina HiSeq 2500 platform to obtain paired-end 100bp reads. .. Reads obtained from parental C1R-CD1d RNA-seq experiments were aligned using TopHat (v2.0.14) ( ) against the Hg19 human reference genome using the default settings.

    Article Title: Thermotolerance in the pathogen Cryptococcus neoformans is linked to antigen masking via mRNA decay-dependent reprogramming
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    Isolation:

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    Article Title: High-throughput Minitaturized RNA-Seq Library Preparation
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    Article Title: Coordinate regulation of alternative pre-mRNA splicing events by the human RNA chaperone proteins hnRNPA1 and DDX5
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    Article Title: Thermotolerance in the pathogen Cryptococcus neoformans is linked to antigen masking via mRNA decay-dependent reprogramming
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    Article Title: Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses
    Article Snippet: .. When targeting eukaryotic mRNA, polyadenylated RNA was isolated using the NEBNext Poly(A) mRNA magnetic isolation module. ..

    Article Title: Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation
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    Article Title: Involvement of condensin in cellular senescence through gene regulation and compartmental reorganization
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    Construct:

    Article Title: Thermotolerance in the pathogen Cryptococcus neoformans is linked to antigen masking via mRNA decay-dependent reprogramming
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    Purification:

    Article Title: Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation
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    Sequencing:

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    New England Biolabs total rna extraction total rna
    Abundance of histone transcripts compared to the remainder of de novo-assembled transcripts in Nanochlorum <t>eucaryotum</t> (SAG 55.87) and five other eukaryotes, including three algae (C. reinhardtii, D. tertiolecta, C. zofingiensis). Abundance of the most highly expressed histone transcript (solid lines) and the summed abundance of all histone transcripts (dashed lines) is indicated for each species. See Methods for details on the underlying <t>RNA-Seq</t> data, assembly and quantification process.
    Total Rna Extraction Total Rna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs monarch total rna miniprep kits
    Abundance of histone transcripts compared to the remainder of de novo-assembled transcripts in Nanochlorum <t>eucaryotum</t> (SAG 55.87) and five other eukaryotes, including three algae (C. reinhardtii, D. tertiolecta, C. zofingiensis). Abundance of the most highly expressed histone transcript (solid lines) and the summed abundance of all histone transcripts (dashed lines) is indicated for each species. See Methods for details on the underlying <t>RNA-Seq</t> data, assembly and quantification process.
    Monarch Total Rna Miniprep Kits, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abundance of histone transcripts compared to the remainder of de novo-assembled transcripts in Nanochlorum eucaryotum (SAG 55.87) and five other eukaryotes, including three algae (C. reinhardtii, D. tertiolecta, C. zofingiensis). Abundance of the most highly expressed histone transcript (solid lines) and the summed abundance of all histone transcripts (dashed lines) is indicated for each species. See Methods for details on the underlying RNA-Seq data, assembly and quantification process.

    Journal: bioRxiv

    Article Title: Slaying the last unicorn - discovery of histones in the microalga Nanochlorum eucaryotum

    doi: 10.1101/2020.10.22.350256

    Figure Lengend Snippet: Abundance of histone transcripts compared to the remainder of de novo-assembled transcripts in Nanochlorum eucaryotum (SAG 55.87) and five other eukaryotes, including three algae (C. reinhardtii, D. tertiolecta, C. zofingiensis). Abundance of the most highly expressed histone transcript (solid lines) and the summed abundance of all histone transcripts (dashed lines) is indicated for each species. See Methods for details on the underlying RNA-Seq data, assembly and quantification process.

    Article Snippet: Total RNA extraction Total RNA was isolated from a 31-day-old N. eucaryotum (SAG 55.87) culture consisting of ~1.9 × 107 cells in total.

    Techniques: RNA Sequencing Assay