luna universal one step rt qpcr kit  (New England Biolabs)


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    New England Biolabs luna universal one step rt qpcr kit
    Screening of compounds with antiviral activity targeting <t>SARS-CoV-2</t> host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by <t>RT-qPCR</t> (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p
    Luna Universal One Step Rt Qpcr Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 39 article reviews
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    1) Product Images from "Characterization and functional interrogation of SARS-CoV-2 RNA interactome"

    Article Title: Characterization and functional interrogation of SARS-CoV-2 RNA interactome

    Journal: bioRxiv

    doi: 10.1101/2021.03.23.436611

    Screening of compounds with antiviral activity targeting SARS-CoV-2 host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p
    Figure Legend Snippet: Screening of compounds with antiviral activity targeting SARS-CoV-2 host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p

    Techniques Used: Activity Assay, Infection, Quantitative RT-PCR

    Functional interrogation of the SARS-CoV-2 RNA interactome and compounds screening. (A) Schematic illustrating the loss-of-function screen procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles released in the supernatant of infected cells was quantified by RT-qPCR and normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data shown are means of two independent experiments. Adjusted p-values were calculated by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors are marked in blue and host restriction factors are marked in red. Positive controls (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained from N protein quantification by flow cytometry and virus release in supernatant of infected cells by RT-qPCR. Data shown are means of two independent experiments. Host dependency factors are marked in blue and host restriction factors are marked in red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown are mean +/- SD of three independent experiments in duplicate.
    Figure Legend Snippet: Functional interrogation of the SARS-CoV-2 RNA interactome and compounds screening. (A) Schematic illustrating the loss-of-function screen procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles released in the supernatant of infected cells was quantified by RT-qPCR and normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data shown are means of two independent experiments. Adjusted p-values were calculated by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors are marked in blue and host restriction factors are marked in red. Positive controls (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained from N protein quantification by flow cytometry and virus release in supernatant of infected cells by RT-qPCR. Data shown are means of two independent experiments. Host dependency factors are marked in blue and host restriction factors are marked in red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown are mean +/- SD of three independent experiments in duplicate.

    Techniques Used: Functional Assay, Transfection, Infection, Quantitative RT-PCR, Flow Cytometry

    2) Product Images from "SARS-CoV-2 Employ BSG/CD147 and ACE2 Receptors to Directly Infect Human Induced Pluripotent Stem Cell-Derived Kidney Podocytes"

    Article Title: SARS-CoV-2 Employ BSG/CD147 and ACE2 Receptors to Directly Infect Human Induced Pluripotent Stem Cell-Derived Kidney Podocytes

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2022.855340

    Susceptibility of human iPS cell-derived podocytes to infection by live SARS-CoV-2. qPCR analysis of human iPS cell-derived podocytes infected with SARS-CoV-2 revealed intracellular uptake of the virus for 24 h.p.i (A) , 48 h.p.i (B) and 72 h.p.i (C) . (D) plaque assay quantification from supernatant obtained from infected podocytes at 24, 48 and 72 h.p.i. (E) qPCR analysis of podocyte-specific genes revealed that both synaptopodin (SYNPO) and podocalyxin (PODXL) are significantly upregulated after infection with SARS-CoV-2 at MOI of 0.01, whereas SYNPO is significantly upregulated at multiple MOIs, and PODXL shows no significant changes with viral infection. (F) The expression of spike-associated genes (ACE2, BSG/CD147) and spike processing genes (TMPRSS2, CTSL) are significantly impacted by infection at MOIs of 0.01 and 0.1, respectively. (G) Human iPS cell-derived podocytes treated with SARS-CoV-2 (at MOI of 0.01) immunostain positive for Nucleocapsid protein (magenta), indicating successful infection with the virus. The cells were immunostained also for the podocyte marker Nephrin (green) and counterstained with DAPI (blue). Scale bar: 100 µm (H) Spike positive cells Nephrin and DAPI as nuclear counterstain in the infected podocytes. Scale bar: 100 µm. One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p
    Figure Legend Snippet: Susceptibility of human iPS cell-derived podocytes to infection by live SARS-CoV-2. qPCR analysis of human iPS cell-derived podocytes infected with SARS-CoV-2 revealed intracellular uptake of the virus for 24 h.p.i (A) , 48 h.p.i (B) and 72 h.p.i (C) . (D) plaque assay quantification from supernatant obtained from infected podocytes at 24, 48 and 72 h.p.i. (E) qPCR analysis of podocyte-specific genes revealed that both synaptopodin (SYNPO) and podocalyxin (PODXL) are significantly upregulated after infection with SARS-CoV-2 at MOI of 0.01, whereas SYNPO is significantly upregulated at multiple MOIs, and PODXL shows no significant changes with viral infection. (F) The expression of spike-associated genes (ACE2, BSG/CD147) and spike processing genes (TMPRSS2, CTSL) are significantly impacted by infection at MOIs of 0.01 and 0.1, respectively. (G) Human iPS cell-derived podocytes treated with SARS-CoV-2 (at MOI of 0.01) immunostain positive for Nucleocapsid protein (magenta), indicating successful infection with the virus. The cells were immunostained also for the podocyte marker Nephrin (green) and counterstained with DAPI (blue). Scale bar: 100 µm (H) Spike positive cells Nephrin and DAPI as nuclear counterstain in the infected podocytes. Scale bar: 100 µm. One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p

    Techniques Used: Derivative Assay, Infection, Real-time Polymerase Chain Reaction, Plaque Assay, Expressing, Marker

    Transcriptomic and protein level analyses of spike-interacting factors in mature podocytes differentiated from human iPS cells. (A) Heatmap showing expression levels of spike associated genes from three biological human iPS cell-derived podocyte samples. SIGLEC9, Sialic acid-binding Ig-like lectin 9; CAPZA1, F-actin-capping protein subunit alpha-1; CLEC10A, C-type lectin domain family 10 member A; CD33, Myeloid cell surface antigen CD33; TMOD3, Tropomodulin-3; ACE2, Angiotensin Converting Enzyme 2; BSG/CD147, Basigin/CD147 molecule; CD209, CD209 Antigen; MYO6, Unconventional myosin-VI; PLS3, Plastin-3; LDHB, L-lactate dehydrogenase B chain; GNB2L1/RACK, Receptor of activated protein C kinase 1; SNRNP70, U1 small nuclear ribonucleoprotein 70 kDa; DOCK7, Dedicator of cytokinesis protein 7; RPS18, 40S ribosomal protein S18; CAPZB, F-actin-capping protein subunit beta; GOLGA7, Golgin subfamily A member 7; ZDHHC5, Palmitoyltransferase ZDHHC5; SIGLEC10, Sialic acid-binding Ig-like lectin 10; ACTR3, Actin-related protein 3; MYL6, Myosin light polypeptide 6; CORO1C, Coronin-1C; ARPC4, Actin-related protein 2/3 complex subunit 4; CCT6A, T-complex protein 1 subunit zeta; (B) qPCR quantification of nine of the human spike-associated gene from (A) including Transmembrane Serine Protease 2 (TMPRSS2) or cathepsin L (CTSL) in human iPS cell-derived podocytes, Calu-3, Caco-2, glomerular endothelial cells, and HEK 293T cells (normalized to Calu-3 groups). (C) Western blot analysis to evaluate protein expression of ACE2, BSG/CD147, CD209, TMPRSS2 and CTSL in the different cell type used; Calu-3, human iPS cell-derived podocytes, Caco-2, glomerular endothelial (gEndos) cells, and HEK 293T cells. (D) Immunocytochemistry analysis of ACE2, BSG/CD147, CD209, TMPRSS2 and CTSL expression in iPS cell-derived podocytes. Scale bar: 100 µm. One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p
    Figure Legend Snippet: Transcriptomic and protein level analyses of spike-interacting factors in mature podocytes differentiated from human iPS cells. (A) Heatmap showing expression levels of spike associated genes from three biological human iPS cell-derived podocyte samples. SIGLEC9, Sialic acid-binding Ig-like lectin 9; CAPZA1, F-actin-capping protein subunit alpha-1; CLEC10A, C-type lectin domain family 10 member A; CD33, Myeloid cell surface antigen CD33; TMOD3, Tropomodulin-3; ACE2, Angiotensin Converting Enzyme 2; BSG/CD147, Basigin/CD147 molecule; CD209, CD209 Antigen; MYO6, Unconventional myosin-VI; PLS3, Plastin-3; LDHB, L-lactate dehydrogenase B chain; GNB2L1/RACK, Receptor of activated protein C kinase 1; SNRNP70, U1 small nuclear ribonucleoprotein 70 kDa; DOCK7, Dedicator of cytokinesis protein 7; RPS18, 40S ribosomal protein S18; CAPZB, F-actin-capping protein subunit beta; GOLGA7, Golgin subfamily A member 7; ZDHHC5, Palmitoyltransferase ZDHHC5; SIGLEC10, Sialic acid-binding Ig-like lectin 10; ACTR3, Actin-related protein 3; MYL6, Myosin light polypeptide 6; CORO1C, Coronin-1C; ARPC4, Actin-related protein 2/3 complex subunit 4; CCT6A, T-complex protein 1 subunit zeta; (B) qPCR quantification of nine of the human spike-associated gene from (A) including Transmembrane Serine Protease 2 (TMPRSS2) or cathepsin L (CTSL) in human iPS cell-derived podocytes, Calu-3, Caco-2, glomerular endothelial cells, and HEK 293T cells (normalized to Calu-3 groups). (C) Western blot analysis to evaluate protein expression of ACE2, BSG/CD147, CD209, TMPRSS2 and CTSL in the different cell type used; Calu-3, human iPS cell-derived podocytes, Caco-2, glomerular endothelial (gEndos) cells, and HEK 293T cells. (D) Immunocytochemistry analysis of ACE2, BSG/CD147, CD209, TMPRSS2 and CTSL expression in iPS cell-derived podocytes. Scale bar: 100 µm. One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p

    Techniques Used: Expressing, Derivative Assay, Binding Assay, Real-time Polymerase Chain Reaction, Western Blot, Immunocytochemistry

    Antibody blocking reveal roles of ACE2 and BSG/CD147 receptors in viral uptake. (A) qPCR quantification of S-pseudotyped entry relative to antibody concentration (normalized to unblocked samples). Human iPS cell-derived podocytes were incubated with different dilutions of anti-ACE2, anti-BSG or both for an hour followed by infection with S-pseudotyped virus at MOI 0.02 for 60 h (B) qPCR quantification of Spike binding receptor genes (ACE2, BSG/CD147) and Spike processing factor genes (TMPRSS2, CTSL) at 5 µg/ml for both anti-ACE2 antibody blockage and anti-BSG antibody blockage showing significant changes in gene expression with optimal receptor blockage when compared to unblocked samples (normalized to mock groups). One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p
    Figure Legend Snippet: Antibody blocking reveal roles of ACE2 and BSG/CD147 receptors in viral uptake. (A) qPCR quantification of S-pseudotyped entry relative to antibody concentration (normalized to unblocked samples). Human iPS cell-derived podocytes were incubated with different dilutions of anti-ACE2, anti-BSG or both for an hour followed by infection with S-pseudotyped virus at MOI 0.02 for 60 h (B) qPCR quantification of Spike binding receptor genes (ACE2, BSG/CD147) and Spike processing factor genes (TMPRSS2, CTSL) at 5 µg/ml for both anti-ACE2 antibody blockage and anti-BSG antibody blockage showing significant changes in gene expression with optimal receptor blockage when compared to unblocked samples (normalized to mock groups). One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p

    Techniques Used: Blocking Assay, Real-time Polymerase Chain Reaction, Concentration Assay, Derivative Assay, Incubation, Infection, Binding Assay, Expressing

    3) Product Images from "Signal Recognition Particle RNA Contributes to Oxidative Stress Response in Deinococcus radiodurans by Modulating Catalase Localization"

    Article Title: Signal Recognition Particle RNA Contributes to Oxidative Stress Response in Deinococcus radiodurans by Modulating Catalase Localization

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.613571

    Knockdown (KD) of Qpr6 leads to the hypersensitivity of D. radiodurans to H 2 O 2 and chronic ionizing radiation (CIR). (A) Evaluation of Qpr6 level in R1, Qpr6 KD, and Qpr6 Com by RT-qPCR; (B) Survival of R1, Qpr6 KD, and Qpr6 Com under H 2 O 2 stress; (C) Growth of R1, Qpr6 KD, and Qpr6 Com under 0 and 57 Gy/h radiation ( γ -ray) for 6 days; (D) Survival rate of R1, Qpr6 KD, and Qpr6 Com under 10 kGy ionizing radiation ( β -ray). Error bars indicate standard errors of the means ( n = 3). ** p ≤ 0.01, and *** p ≤ 0.001 were considered as significant using the Student t -test, while ns indicates non-significant compared to R1 ( p > 0.05).
    Figure Legend Snippet: Knockdown (KD) of Qpr6 leads to the hypersensitivity of D. radiodurans to H 2 O 2 and chronic ionizing radiation (CIR). (A) Evaluation of Qpr6 level in R1, Qpr6 KD, and Qpr6 Com by RT-qPCR; (B) Survival of R1, Qpr6 KD, and Qpr6 Com under H 2 O 2 stress; (C) Growth of R1, Qpr6 KD, and Qpr6 Com under 0 and 57 Gy/h radiation ( γ -ray) for 6 days; (D) Survival rate of R1, Qpr6 KD, and Qpr6 Com under 10 kGy ionizing radiation ( β -ray). Error bars indicate standard errors of the means ( n = 3). ** p ≤ 0.01, and *** p ≤ 0.001 were considered as significant using the Student t -test, while ns indicates non-significant compared to R1 ( p > 0.05).

    Techniques Used: Quantitative RT-PCR

    4) Product Images from "LncRNA TUG1 was upregulated in osteoporosis and regulates the proliferation and apoptosis of osteoclasts"

    Article Title: LncRNA TUG1 was upregulated in osteoporosis and regulates the proliferation and apoptosis of osteoclasts

    Journal: Journal of Orthopaedic Surgery and Research

    doi: 10.1186/s13018-019-1430-4

    Plasma lncRNA TUG1 was upregulated in osteoporosis patients than in healthy participants. RT-qPCR results showed that plasma levels of lncRNA TUG1 were significantly higher in osteoporosis patients than in healthy participants (* p
    Figure Legend Snippet: Plasma lncRNA TUG1 was upregulated in osteoporosis patients than in healthy participants. RT-qPCR results showed that plasma levels of lncRNA TUG1 were significantly higher in osteoporosis patients than in healthy participants (* p

    Techniques Used: Quantitative RT-PCR

    5) Product Images from "Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation"

    Article Title: Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation

    Journal: eLife

    doi: 10.7554/eLife.54655

    ArcZ can influence CyaR levels. ( A ) Base-pairing interactions predicted from the ArcZ-CyaR chimeras using RNACofold. The nucleotide substitutions for experimental validation of direct base-pairing are shown as red or green residues. ( B ) Northern blot analysis of ArcZ and CyaR. The cells containing both the empty pZA and pJV300 plasmids (lanes 1, 5, 7) do not express ArcZ and CyaR at detectable levels. ( C ) Validation of ArcZ-CyaR interaction by over-expression analyses. ArcZ (panel I) orCyaR (panel II) was over-expressed and the levels of their targets were monitored by RT-qPCR. The tpx and sdaC mRNAs are ArcZ targets (panel I). The nadE and yqaE mRNAs are CyaR targets (panel II). The dashed horizontal line indicates the level in the control plasmid (pJV300) that expresses a ~50 nt randomly generated RNA sequence. Panel III: The sRNAs and mutants (as in (A)) were ectopically co-expressed in E. coli and CyaR and CyaR 38–39 levels were quantified by RT-qPCR. Experiments were performed in biological and technical triplicates; Error bars indicate the standard error of the mean (SEM) of the three biological replicates. ( D ) ArcZ and CyaR were overexpressed from a plasmid-borne IPTG inducible promoter (pZE-ArcZ and pZE-CyaR) and the data were compared to data from cells carrying plasmid pJV300. The co-expressed candidate target sRNAs (expressed from pZA-derived backbone) were induced with anhydrotetracycline hydrochloride (panels I and II). The bars indicate the mean fold-change in expression relative to the level of 5S rRNA ( rrfD ) in cells with the indicated vector. In panel III endogenous ArcZ levels were measured upon over-expression of CyaR. Error bars indicate the standard error of the mean from three biological replicates and three technical replicates per experiment. Source data are provided as a Source Data file. Source data for Figure 7B . Source data for Figure 7C . Source data for Figure 7D .
    Figure Legend Snippet: ArcZ can influence CyaR levels. ( A ) Base-pairing interactions predicted from the ArcZ-CyaR chimeras using RNACofold. The nucleotide substitutions for experimental validation of direct base-pairing are shown as red or green residues. ( B ) Northern blot analysis of ArcZ and CyaR. The cells containing both the empty pZA and pJV300 plasmids (lanes 1, 5, 7) do not express ArcZ and CyaR at detectable levels. ( C ) Validation of ArcZ-CyaR interaction by over-expression analyses. ArcZ (panel I) orCyaR (panel II) was over-expressed and the levels of their targets were monitored by RT-qPCR. The tpx and sdaC mRNAs are ArcZ targets (panel I). The nadE and yqaE mRNAs are CyaR targets (panel II). The dashed horizontal line indicates the level in the control plasmid (pJV300) that expresses a ~50 nt randomly generated RNA sequence. Panel III: The sRNAs and mutants (as in (A)) were ectopically co-expressed in E. coli and CyaR and CyaR 38–39 levels were quantified by RT-qPCR. Experiments were performed in biological and technical triplicates; Error bars indicate the standard error of the mean (SEM) of the three biological replicates. ( D ) ArcZ and CyaR were overexpressed from a plasmid-borne IPTG inducible promoter (pZE-ArcZ and pZE-CyaR) and the data were compared to data from cells carrying plasmid pJV300. The co-expressed candidate target sRNAs (expressed from pZA-derived backbone) were induced with anhydrotetracycline hydrochloride (panels I and II). The bars indicate the mean fold-change in expression relative to the level of 5S rRNA ( rrfD ) in cells with the indicated vector. In panel III endogenous ArcZ levels were measured upon over-expression of CyaR. Error bars indicate the standard error of the mean from three biological replicates and three technical replicates per experiment. Source data are provided as a Source Data file. Source data for Figure 7B . Source data for Figure 7C . Source data for Figure 7D .

    Techniques Used: Northern Blot, Over Expression, Quantitative RT-PCR, Plasmid Preparation, Generated, Sequencing, Derivative Assay, Expressing

    6) Product Images from "SARS-CoV-2 and SARS-CoV differ in their cell tropism and drug sensitivity profiles"

    Article Title: SARS-CoV-2 and SARS-CoV differ in their cell tropism and drug sensitivity profiles

    Journal: bioRxiv

    doi: 10.1101/2020.04.03.024257

    N A) Western blots indicating cellular ACE2 and TMPRSS2 protein levels. B) CPE formation in SARS-CoV and SARS-CoV-2 (MOI 0.01)-infected ACE2-negative 293 cells and 293 cells stably expressing ACE2 cells (293/ACE2) 48h post infection. C) Immunostaining for double-stranded RNA in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected 293/ACE2 cells 48h post infection. D) Quantification of virus genomes by qPCR in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected 293/ACE2 cells 48h post infection. E) Cytopathogenic effect (CPE) formation in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected Caco2 cells in the presence of antibodies directed against ACE2 or DPP4 (MERS-CoV receptor) 48h post infection.
    Figure Legend Snippet: N A) Western blots indicating cellular ACE2 and TMPRSS2 protein levels. B) CPE formation in SARS-CoV and SARS-CoV-2 (MOI 0.01)-infected ACE2-negative 293 cells and 293 cells stably expressing ACE2 cells (293/ACE2) 48h post infection. C) Immunostaining for double-stranded RNA in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected 293/ACE2 cells 48h post infection. D) Quantification of virus genomes by qPCR in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected 293/ACE2 cells 48h post infection. E) Cytopathogenic effect (CPE) formation in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected Caco2 cells in the presence of antibodies directed against ACE2 or DPP4 (MERS-CoV receptor) 48h post infection.

    Techniques Used: Western Blot, Infection, Stable Transfection, Expressing, Immunostaining, Real-time Polymerase Chain Reaction

    SARS-CoV-2 and SARS-CoV susceptibility of colorectal cancer cell lines. A) Cytopathogenic effect (CPE) formation 48h post infection in MOI 0.01-infected cells. B) Representative images showing MOI 0.01-infected cells immunostained for double-stranded RNA 48h post infection. C) Quantification of virus genomes by qPCR at different time points post infection (p.i.).
    Figure Legend Snippet: SARS-CoV-2 and SARS-CoV susceptibility of colorectal cancer cell lines. A) Cytopathogenic effect (CPE) formation 48h post infection in MOI 0.01-infected cells. B) Representative images showing MOI 0.01-infected cells immunostained for double-stranded RNA 48h post infection. C) Quantification of virus genomes by qPCR at different time points post infection (p.i.).

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    7) Product Images from "The barley leaf rust resistance gene Rph3 encodes a predicted membrane protein and is induced upon infection by avirulent pathotypes of Puccinia hordei"

    Article Title: The barley leaf rust resistance gene Rph3 encodes a predicted membrane protein and is induced upon infection by avirulent pathotypes of Puccinia hordei

    Journal: Nature Communications

    doi: 10.1038/s41467-022-29840-1

    Transcript levels of Rph3 detected by RT-qPCR during 2–12 dpi in response to virulent and avirulent pathotypes. The Rph3 gene was upregulated when the leaf was infected by Rph3 -avirulent P. hordei pathotypes (green dot = 200 P−, green square = 5453 P+), whereas the transcript levels were unchanged when the leaf was infected by Rph3 -virulent pathotypes (yellow cross = 5656 P+, yellow asterisk = 5457 P+) and P. triticina (blue diamond = 26-0, blue triangle = 104-1,2,3,(6),(7),11,13). The transcript levels of Rph3 in un-inoculated seedlings (mock inoculation) is shown in the black line. Values represent means ± SE (shown as the error bars) ( n = 3). Samples inoculated with pathotype 5453 P+ at two dpi were used as calibrations to calculate the relative quantification (RQ) values using the delta-delta method with RQ = 2 −∆∆Cq . The ADP-ribosylation factor gene was used as a normalizer.
    Figure Legend Snippet: Transcript levels of Rph3 detected by RT-qPCR during 2–12 dpi in response to virulent and avirulent pathotypes. The Rph3 gene was upregulated when the leaf was infected by Rph3 -avirulent P. hordei pathotypes (green dot = 200 P−, green square = 5453 P+), whereas the transcript levels were unchanged when the leaf was infected by Rph3 -virulent pathotypes (yellow cross = 5656 P+, yellow asterisk = 5457 P+) and P. triticina (blue diamond = 26-0, blue triangle = 104-1,2,3,(6),(7),11,13). The transcript levels of Rph3 in un-inoculated seedlings (mock inoculation) is shown in the black line. Values represent means ± SE (shown as the error bars) ( n = 3). Samples inoculated with pathotype 5453 P+ at two dpi were used as calibrations to calculate the relative quantification (RQ) values using the delta-delta method with RQ = 2 −∆∆Cq . The ADP-ribosylation factor gene was used as a normalizer.

    Techniques Used: Quantitative RT-PCR, Infection

    8) Product Images from "Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen"

    Article Title: Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen

    Journal: Nature Communications

    doi: 10.1038/s41467-022-30134-9

    DAXX restriction of SARS-CoV-2 is dependent on its chaperone activity but SUMOylation-independent. a Schematic of the DAXX mutants used. The fifteen lysine residues of DAXX 15KR have been mutated to arginine. DAXX∆SIM lacks the 732–740 C-terminal residues. Both mutants were described in. 48 DAXX∆D/E is lacking its 414-505 domain and has been described in 52 b , c SUMOylation-deficient DAXX mutants are still able to restrict SARS-CoV-2. 293T-ACE2 cells were transfected with HA-DAXX WT; HA-DAXX 15KR; HA-DAXX∆SIM; or with HA-NBR1 as negative control plasmid. 24 h after transfection, cells were infected with SARS-CoV-2 at an MOI of 0.1. When indicated, cells were treated with remdesivir at the time of infection. After 24 h or 48 h, infected cells were double-stained for dsRNA (to read out infection) and HA (to read out transfection efficiency) and acquired by flow cytometry. The percentage of infected cells among HA-positive (transfected) cells for one representative experiment is shown in ( b ), for the mean ± SD of 3 independent experiments in ( c ). Statistics: one-way ANOVA using Dunnett’s test, Holm corrected. P values are indicated on the graph. d , e : The chaperone activity of DAXX is required for SARS-CoV-2 restriction. 293T-ACE2 cells were transfected with DAXX WT or with the DAXXΔD/E mutant. 24 h after transfection, cells were infected with SARS-CoV-2 mNeonGreen at an MOI of 1. After 24 h, the levels of Spike and GAPDH levels were analyzed by Western Blot ( d ). A Western Blot representative of 3 independent experiments is shown. In parallel, SARS-CoV-2 replication levels were measured by RT-qPCR targeting the 5ʹ UTR and normalized against RPL13A transcript levels ( e ). The mean ± SD of 4 independent experiments, with infections carried out in two biological replicates, is represented. Statistics: 1-way ANOVA using Dunnett’s test. P values are indicated on the graph. Source data are provided as a Source Data file.
    Figure Legend Snippet: DAXX restriction of SARS-CoV-2 is dependent on its chaperone activity but SUMOylation-independent. a Schematic of the DAXX mutants used. The fifteen lysine residues of DAXX 15KR have been mutated to arginine. DAXX∆SIM lacks the 732–740 C-terminal residues. Both mutants were described in. 48 DAXX∆D/E is lacking its 414-505 domain and has been described in 52 b , c SUMOylation-deficient DAXX mutants are still able to restrict SARS-CoV-2. 293T-ACE2 cells were transfected with HA-DAXX WT; HA-DAXX 15KR; HA-DAXX∆SIM; or with HA-NBR1 as negative control plasmid. 24 h after transfection, cells were infected with SARS-CoV-2 at an MOI of 0.1. When indicated, cells were treated with remdesivir at the time of infection. After 24 h or 48 h, infected cells were double-stained for dsRNA (to read out infection) and HA (to read out transfection efficiency) and acquired by flow cytometry. The percentage of infected cells among HA-positive (transfected) cells for one representative experiment is shown in ( b ), for the mean ± SD of 3 independent experiments in ( c ). Statistics: one-way ANOVA using Dunnett’s test, Holm corrected. P values are indicated on the graph. d , e : The chaperone activity of DAXX is required for SARS-CoV-2 restriction. 293T-ACE2 cells were transfected with DAXX WT or with the DAXXΔD/E mutant. 24 h after transfection, cells were infected with SARS-CoV-2 mNeonGreen at an MOI of 1. After 24 h, the levels of Spike and GAPDH levels were analyzed by Western Blot ( d ). A Western Blot representative of 3 independent experiments is shown. In parallel, SARS-CoV-2 replication levels were measured by RT-qPCR targeting the 5ʹ UTR and normalized against RPL13A transcript levels ( e ). The mean ± SD of 4 independent experiments, with infections carried out in two biological replicates, is represented. Statistics: 1-way ANOVA using Dunnett’s test. P values are indicated on the graph. Source data are provided as a Source Data file.

    Techniques Used: Activity Assay, Transfection, Negative Control, Plasmid Preparation, Infection, Staining, Flow Cytometry, Mutagenesis, Western Blot, Quantitative RT-PCR

    9) Product Images from "Sublethal enteroviral infection exacerbates disease progression in an ALS mouse model"

    Article Title: Sublethal enteroviral infection exacerbates disease progression in an ALS mouse model

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-022-02380-7

    CVB3-accelerated ALS progression is mitigated by early antiviral treatment. A Schematic diagram illustrating the experimental plan for CVB3 injection, ribavirin administration, and the endpoint. B Kaplan–Meier plots comparing mouse survivals among SOD1 G85R CVB3 ( n = 8; male = 2 and female = 6), SOD1 G85R CVB3 day 1 ribavirin ( n = 5; male = 2 and female = 3), and SOD1 G85R CVB3 day 15 ribavirin ( n = 5; male = 2 and female = 3) mice. P = 0.0037 between SOD1 G85R CVB3 and SOD1 G85R CVB3 day 1 ribavirin groups. C Mouse motor function measured weekly (i.e., hindlimb reflex score, inverted grid time, footprint time, and stride distance) starting at week 20 PI until humane or experimental endpoint. The vertical black line indicates the point of deviation between the two groups. D – E RT-qPCR evaluation of proinflammatory genes ( D ) and viral RNA ( E ) in the brain tissues collected at week 60 PI or the humane endpoints. F Representative TDP-43 IHC staining images in the hippocampus regions of the brain collected at week 60 PI or the humane endpoint. The red arrows indicate TDP-43 mislocalization. Scale bar = 100 μm. Quantifications are presented as mean ± SEM ( n = 3–4). Statistical analysis was carried out by two-way ANOVA, followed by Bonferroni’s multiple comparison test. *, p
    Figure Legend Snippet: CVB3-accelerated ALS progression is mitigated by early antiviral treatment. A Schematic diagram illustrating the experimental plan for CVB3 injection, ribavirin administration, and the endpoint. B Kaplan–Meier plots comparing mouse survivals among SOD1 G85R CVB3 ( n = 8; male = 2 and female = 6), SOD1 G85R CVB3 day 1 ribavirin ( n = 5; male = 2 and female = 3), and SOD1 G85R CVB3 day 15 ribavirin ( n = 5; male = 2 and female = 3) mice. P = 0.0037 between SOD1 G85R CVB3 and SOD1 G85R CVB3 day 1 ribavirin groups. C Mouse motor function measured weekly (i.e., hindlimb reflex score, inverted grid time, footprint time, and stride distance) starting at week 20 PI until humane or experimental endpoint. The vertical black line indicates the point of deviation between the two groups. D – E RT-qPCR evaluation of proinflammatory genes ( D ) and viral RNA ( E ) in the brain tissues collected at week 60 PI or the humane endpoints. F Representative TDP-43 IHC staining images in the hippocampus regions of the brain collected at week 60 PI or the humane endpoint. The red arrows indicate TDP-43 mislocalization. Scale bar = 100 μm. Quantifications are presented as mean ± SEM ( n = 3–4). Statistical analysis was carried out by two-way ANOVA, followed by Bonferroni’s multiple comparison test. *, p

    Techniques Used: Injection, Mouse Assay, Quantitative RT-PCR, Immunohistochemistry, Staining

    10) Product Images from "Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry"

    Article Title: Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

    Journal: bioRxiv

    doi: 10.1101/2020.10.05.326082

    Determination of RNA-compatible fixation and permeabilisation conditions A : 1×10 6 COLO205 cells were either unfixed (lane 1), fixed with 70% ethanol on ice for 15 minutes (lane 2) or fixed with 4% formaldehyde on ice for 15 minutes (lane 3). Unfixed cells were dissolved immediately in TRI Reagent, fixed cells were washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C before RNA extraction with TRI Reagent. 20% of RNA obtained was separated on a 1.2% glyoxal gel and imaged by ethidium bromide staining. B : COLO205 cells were either unfixed (lane 1), fixed with 70% ethanol on ice for 15 minutes (lane 2) or fixed with 100% methanol on ice for 15 minutes (lane 3). Unfixed cells were dissolved immediately in TRI Reagent, fixed cells were washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C before RNA extraction with TRI Reagent. RNA was analysed as in A . C : COLO205 cells were fixed with glyoxal fixation mix (pH4) either without or with 20% ethanol and incubated on ice for 15 minutes and washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C. RNA was extracted and analysed as in A . D : COLO205 cells were either unfixed (lane 1), fixed with glyoxal fixation mix (pH4) with 20% ethanol (lane 2) or with 4% formaldehyde on ice for 15 minutes (lane 3). Cells were washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C and incubated on ice for 1 hour in 100 μl PBS followed by centrifugation at 2000 x g for 3 minutes at 4°C before RNA extraction with an RNeasy mini kit. E : 100 ng RNA per reaction from D was subjected to one-step combined reverse transcription and quantitative PCR reactions for ACT1B, GAPDH and PGK1. Ct is the cycle number at which the fluorescence exceeded threshold. 3 technical replicates for each RT-qPCR reaction were performed. F : COLO205 cells were either unfixed (lane 1) or fixed with glyoxal fixation mix (pH4) with 20% ethanol on ice for 15 minutes and permeabilised in 100% methanol on ice for 30 minutes (lane 2), or 0.5% saponin on ice for 30 minutes (lane 3) or 0.3% Triton X-100 on ice for 30 minutes (lane 4). RNA was analysed as in A . G : COLO205 cells were either unfixed (lane 1) or fixed with glyoxal fixation mix (pH4) with 20% ethanol on ice for 15 minutes, permeabilised in 100% methanol on ice for 30 minutes followed by incubation in primary antibody for 1 hour on ice and secondary antibody for 30 minutes on ice in dark (lane 2). RNA was analysed as in A .
    Figure Legend Snippet: Determination of RNA-compatible fixation and permeabilisation conditions A : 1×10 6 COLO205 cells were either unfixed (lane 1), fixed with 70% ethanol on ice for 15 minutes (lane 2) or fixed with 4% formaldehyde on ice for 15 minutes (lane 3). Unfixed cells were dissolved immediately in TRI Reagent, fixed cells were washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C before RNA extraction with TRI Reagent. 20% of RNA obtained was separated on a 1.2% glyoxal gel and imaged by ethidium bromide staining. B : COLO205 cells were either unfixed (lane 1), fixed with 70% ethanol on ice for 15 minutes (lane 2) or fixed with 100% methanol on ice for 15 minutes (lane 3). Unfixed cells were dissolved immediately in TRI Reagent, fixed cells were washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C before RNA extraction with TRI Reagent. RNA was analysed as in A . C : COLO205 cells were fixed with glyoxal fixation mix (pH4) either without or with 20% ethanol and incubated on ice for 15 minutes and washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C. RNA was extracted and analysed as in A . D : COLO205 cells were either unfixed (lane 1), fixed with glyoxal fixation mix (pH4) with 20% ethanol (lane 2) or with 4% formaldehyde on ice for 15 minutes (lane 3). Cells were washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C and incubated on ice for 1 hour in 100 μl PBS followed by centrifugation at 2000 x g for 3 minutes at 4°C before RNA extraction with an RNeasy mini kit. E : 100 ng RNA per reaction from D was subjected to one-step combined reverse transcription and quantitative PCR reactions for ACT1B, GAPDH and PGK1. Ct is the cycle number at which the fluorescence exceeded threshold. 3 technical replicates for each RT-qPCR reaction were performed. F : COLO205 cells were either unfixed (lane 1) or fixed with glyoxal fixation mix (pH4) with 20% ethanol on ice for 15 minutes and permeabilised in 100% methanol on ice for 30 minutes (lane 2), or 0.5% saponin on ice for 30 minutes (lane 3) or 0.3% Triton X-100 on ice for 30 minutes (lane 4). RNA was analysed as in A . G : COLO205 cells were either unfixed (lane 1) or fixed with glyoxal fixation mix (pH4) with 20% ethanol on ice for 15 minutes, permeabilised in 100% methanol on ice for 30 minutes followed by incubation in primary antibody for 1 hour on ice and secondary antibody for 30 minutes on ice in dark (lane 2). RNA was analysed as in A .

    Techniques Used: Centrifugation, RNA Extraction, Staining, Incubation, Real-time Polymerase Chain Reaction, Fluorescence, Quantitative RT-PCR

    11) Product Images from "Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition"

    Article Title: Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2022.111117

    A6-001, an ANO6 inhibitor, inhibits the viral replication of SARS-CoV-2 in Calu-3, Vero, and human nasal epithelial (HNE) cells (A and B) Viral replication of authentic SARS-CoV-2 (0.001 MOI) was assayed in Calu-3 cells. The qPCR results of virion mRNAs with the indicated concentrations of A6-001 are shown in (A) ( ∗ p
    Figure Legend Snippet: A6-001, an ANO6 inhibitor, inhibits the viral replication of SARS-CoV-2 in Calu-3, Vero, and human nasal epithelial (HNE) cells (A and B) Viral replication of authentic SARS-CoV-2 (0.001 MOI) was assayed in Calu-3 cells. The qPCR results of virion mRNAs with the indicated concentrations of A6-001 are shown in (A) ( ∗ p

    Techniques Used: Real-time Polymerase Chain Reaction

    12) Product Images from "Dissecting transcriptional amplification by MYC"

    Article Title: Dissecting transcriptional amplification by MYC

    Journal: eLife

    doi: 10.7554/eLife.52483

    MYC-Box mutations change both non-E and E-box promoter-output at the RNA level. ( A and B ) Cells were transfected as noted with 80 ng of empty vector, MYC, MBII or MBIII expressing vectors, 100 ng of non-E-box or E-box reporter and 2 ng of GR plasmids . Luciferase and renilla luciferase were assayed. ( C ). Transfected cells as in B were harvested and RNA extracted for RT-qPCR using Luna Universal One-Step RT-qPCR Kit (New England Biolabs). Experiments were performed in biological triplicate. Standard deviations are indicated. *,** and *** indicate p≤0.05, 0.01, 0.001, respectively, one-tailed t-test. Experiments performed in triplicate (A and B, n = 3; C, n = 1).
    Figure Legend Snippet: MYC-Box mutations change both non-E and E-box promoter-output at the RNA level. ( A and B ) Cells were transfected as noted with 80 ng of empty vector, MYC, MBII or MBIII expressing vectors, 100 ng of non-E-box or E-box reporter and 2 ng of GR plasmids . Luciferase and renilla luciferase were assayed. ( C ). Transfected cells as in B were harvested and RNA extracted for RT-qPCR using Luna Universal One-Step RT-qPCR Kit (New England Biolabs). Experiments were performed in biological triplicate. Standard deviations are indicated. *,** and *** indicate p≤0.05, 0.01, 0.001, respectively, one-tailed t-test. Experiments performed in triplicate (A and B, n = 3; C, n = 1).

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Luciferase, Quantitative RT-PCR, One-tailed Test

    MYC-EGFP binding at chromosomally integrated-reporters parallels activity and emulates native promoters. Top: diagram of integrated-lentivirus CFP reporters. ( A ) Left-ChIP-PCR to assay MYC-EGFP binding in the absence of activator of uninduced (lanes 2 and 6) versus doxycycline-induced (lanes 4 and 8) MYC-EGFP, at non-E-box (lanes 2 and 4) versus E-box-bearing (lanes 6 and 8) lentivirus-integrated reporter promoters. Right-ChIP-PCR to assay MYC-EGFP binding in the presence of GAL4/VP16 of uninduced (lanes 10 and 14) versus doxycycline-induced (lanes 12 and 16) MYC-EGFP at non-E-box (lanes 10 and 12) versus E-box bearing (lanes 14 and 16) lentivirus integrated reporter-promoters. ( B ) EZH2 RNA levels from cells expressing non-induced or doxycycline induced MYC-EGFP(WT) or MYC-EGFP-mutants MBI, MBII, MBIII or MBIV and assayed by RT-qPCR using Luna Universal One-Step RT-qPCR Kit (New England Biolabs). ( C ) Binding of uninduced and induced MYC-EGFP to endogenous promoters. The asterisk (*) indicates that binding to BEX in heterochromatin was so low as to be virtually unexpressed as previously reported ( Nie et al., 2012 ). ( D ). Uninduced or doxycycline-induced MYC-EGFP(WT) or MYC-EGFP-mutants (MBI, MBII, MBIII, or MBIV) binding to the native EZH2 promoter assayed by ChIP-PCR. The mean and SD for representative experiments performed in triplicate are shown, n ≥ 2.
    Figure Legend Snippet: MYC-EGFP binding at chromosomally integrated-reporters parallels activity and emulates native promoters. Top: diagram of integrated-lentivirus CFP reporters. ( A ) Left-ChIP-PCR to assay MYC-EGFP binding in the absence of activator of uninduced (lanes 2 and 6) versus doxycycline-induced (lanes 4 and 8) MYC-EGFP, at non-E-box (lanes 2 and 4) versus E-box-bearing (lanes 6 and 8) lentivirus-integrated reporter promoters. Right-ChIP-PCR to assay MYC-EGFP binding in the presence of GAL4/VP16 of uninduced (lanes 10 and 14) versus doxycycline-induced (lanes 12 and 16) MYC-EGFP at non-E-box (lanes 10 and 12) versus E-box bearing (lanes 14 and 16) lentivirus integrated reporter-promoters. ( B ) EZH2 RNA levels from cells expressing non-induced or doxycycline induced MYC-EGFP(WT) or MYC-EGFP-mutants MBI, MBII, MBIII or MBIV and assayed by RT-qPCR using Luna Universal One-Step RT-qPCR Kit (New England Biolabs). ( C ) Binding of uninduced and induced MYC-EGFP to endogenous promoters. The asterisk (*) indicates that binding to BEX in heterochromatin was so low as to be virtually unexpressed as previously reported ( Nie et al., 2012 ). ( D ). Uninduced or doxycycline-induced MYC-EGFP(WT) or MYC-EGFP-mutants (MBI, MBII, MBIII, or MBIV) binding to the native EZH2 promoter assayed by ChIP-PCR. The mean and SD for representative experiments performed in triplicate are shown, n ≥ 2.

    Techniques Used: Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

    13) Product Images from "A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing"

    Article Title: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

    Journal: bioRxiv

    doi: 10.1101/2020.06.23.166397

    A sensitive, robust RT-LAMP assay compatible with crude patient samples. A) Schematic illustrating loop-mediated amplification (LAMP) of SARS-CoV-2 RNA and the regions targeted in this study (Orf1ab, E and N genes; depicted above). Each target region is recognized by a defined set of primers (B3, LB, BIP, LF, FIP, F3). The RNA template (red) is reverse transcribed and displaced after first-strand synthesis; the outer primer binding sites are added in the subsequent amplification step. The resulting dumbbell DNA structure acts as template for further rounds of amplification, ultimately leading to high molecular weight amplicons. B) Readout of a real-time fluorescence RT-LAMP reaction using 500 copies of synthetic SARS-CoV-2 (red) or water as non-targeting control (NTC, black) as input. ‘Time to threshold’ indicates the time at which the fluorescence value reaches threshold level (equivalent to Cq value in RT-qPCR assays), ‘end-point RFU’ indicates the fluorescence value (FAM filter set, absorption/emission at 494 nm/518 nm) after 35 minutes reaction time (used throughout this study unless indicated otherwise); RFU: relative fluorescence units. C) Performance of the three top primer sets for RT-LAMP-based SARS-CoV-2 detection. End-point relative fluorescence units (RFUs) of RT-LAMP reactions (in duplicates) using the indicated primer sets and serially diluted synthetic SARS-CoV-2 RNA standard as input. Water was used as no-target control (NTC). D) Cartoon indicating the workflow for SARS-CoV-2 detection by either RT-LAMP or 1-step RT-qPCR from patient samples (nasopharyngeal swab or gargle) with prior RNA isolation. E) Comparison of RT-LAMP and RT-qPCR performance. Plotted are RT-LAMP end-point fluorescence values after 35 minutes versus the respective RT-qPCR Cq values. RNA was derived from gargle (green) or nasopharyngeal swabs (black); two no-target controls were included (black cross). Reactions in which no amplification was recorded are labelled as qPCR negative. F) Predictive agreement between RT-LAMP and 1-step RT-qPCR assays. Shown are percentages of positive (detected in RT-LAMP and RT-qPCR, black bars) and negative (not detected in either RT-LAMP or RT-qPCR, purple bars) predictive agreement for sample groups (defined by RT-qPCR-derived Cq values) between RT-LAMP (using E- and/or N-gene primers) and 1-step RT-qPCR. G) Performance of different crude sample preparation methods in RT-LAMP. Shown are end-point relative fluorescence units (RFUs) for RT-LAMP reactions targeting human RNAseP on sample inputs derived from defined numbers of HEK293 cells mixed 1:1 with indicated 2x buffers (extracted RNsA served as a positive control). H) Cartoon indicating the workflow for RT-LAMP using QuickExtract crude lysate as sample input. I) Comparison of QuickExtract crude sample input versus extracted RNA as input using 1-step RT-qPCR. Covid-19 patient nasopharyngeal swabs or gargle samples (color coded according to the indicated collection medium) were either processed with the QuickExtract workflow (crude sample input) or RNA was extracted using an automated King Fisher RNA bead purification protocol. Reactions in which no amplification was recorded are labelled as qPCR negative. J) Performance of RT-LAMP with QuickExtract treated crude Covid-19 patient sample input (same samples as in I). Depicted is the correlation of Cq values from RT-qPCR performed on QuickExtract treated samples versus corresponding end-point relative fluorescence units (RFUs) from RT-LAMP reactions.
    Figure Legend Snippet: A sensitive, robust RT-LAMP assay compatible with crude patient samples. A) Schematic illustrating loop-mediated amplification (LAMP) of SARS-CoV-2 RNA and the regions targeted in this study (Orf1ab, E and N genes; depicted above). Each target region is recognized by a defined set of primers (B3, LB, BIP, LF, FIP, F3). The RNA template (red) is reverse transcribed and displaced after first-strand synthesis; the outer primer binding sites are added in the subsequent amplification step. The resulting dumbbell DNA structure acts as template for further rounds of amplification, ultimately leading to high molecular weight amplicons. B) Readout of a real-time fluorescence RT-LAMP reaction using 500 copies of synthetic SARS-CoV-2 (red) or water as non-targeting control (NTC, black) as input. ‘Time to threshold’ indicates the time at which the fluorescence value reaches threshold level (equivalent to Cq value in RT-qPCR assays), ‘end-point RFU’ indicates the fluorescence value (FAM filter set, absorption/emission at 494 nm/518 nm) after 35 minutes reaction time (used throughout this study unless indicated otherwise); RFU: relative fluorescence units. C) Performance of the three top primer sets for RT-LAMP-based SARS-CoV-2 detection. End-point relative fluorescence units (RFUs) of RT-LAMP reactions (in duplicates) using the indicated primer sets and serially diluted synthetic SARS-CoV-2 RNA standard as input. Water was used as no-target control (NTC). D) Cartoon indicating the workflow for SARS-CoV-2 detection by either RT-LAMP or 1-step RT-qPCR from patient samples (nasopharyngeal swab or gargle) with prior RNA isolation. E) Comparison of RT-LAMP and RT-qPCR performance. Plotted are RT-LAMP end-point fluorescence values after 35 minutes versus the respective RT-qPCR Cq values. RNA was derived from gargle (green) or nasopharyngeal swabs (black); two no-target controls were included (black cross). Reactions in which no amplification was recorded are labelled as qPCR negative. F) Predictive agreement between RT-LAMP and 1-step RT-qPCR assays. Shown are percentages of positive (detected in RT-LAMP and RT-qPCR, black bars) and negative (not detected in either RT-LAMP or RT-qPCR, purple bars) predictive agreement for sample groups (defined by RT-qPCR-derived Cq values) between RT-LAMP (using E- and/or N-gene primers) and 1-step RT-qPCR. G) Performance of different crude sample preparation methods in RT-LAMP. Shown are end-point relative fluorescence units (RFUs) for RT-LAMP reactions targeting human RNAseP on sample inputs derived from defined numbers of HEK293 cells mixed 1:1 with indicated 2x buffers (extracted RNsA served as a positive control). H) Cartoon indicating the workflow for RT-LAMP using QuickExtract crude lysate as sample input. I) Comparison of QuickExtract crude sample input versus extracted RNA as input using 1-step RT-qPCR. Covid-19 patient nasopharyngeal swabs or gargle samples (color coded according to the indicated collection medium) were either processed with the QuickExtract workflow (crude sample input) or RNA was extracted using an automated King Fisher RNA bead purification protocol. Reactions in which no amplification was recorded are labelled as qPCR negative. J) Performance of RT-LAMP with QuickExtract treated crude Covid-19 patient sample input (same samples as in I). Depicted is the correlation of Cq values from RT-qPCR performed on QuickExtract treated samples versus corresponding end-point relative fluorescence units (RFUs) from RT-LAMP reactions.

    Techniques Used: RT Lamp Assay, Amplification, Binding Assay, Molecular Weight, Fluorescence, Quantitative RT-PCR, Isolation, Derivative Assay, Real-time Polymerase Chain Reaction, Sample Prep, Positive Control, Purification

    14) Product Images from "LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples"

    Article Title: LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0258263

    Implementation and adaptation of the Saliva Direct RT-qPCR assay to detect SARS-CoV-2 N1 and human RNase P with the QuantStudio-6. a) Full and half reaction volumes on the CFx96 with 2x master mix, b) Full and half reaction volumes on the QuantStudio-6 with 2x master mix, c) Full and half reaction volumes on the QuantStudio-6 with 4x master mix. Samples which failed to amplify are denoted as “not detected” (ND). Plasmid DNA was added directly into the master mix at 200,000 copies/mL (2-4x Saliva Direct limit of detection) in triplicate. Full reactions (15μL master mix with 5μL sample input), half reactions (7.5μL master mix with 5μL sample input).
    Figure Legend Snippet: Implementation and adaptation of the Saliva Direct RT-qPCR assay to detect SARS-CoV-2 N1 and human RNase P with the QuantStudio-6. a) Full and half reaction volumes on the CFx96 with 2x master mix, b) Full and half reaction volumes on the QuantStudio-6 with 2x master mix, c) Full and half reaction volumes on the QuantStudio-6 with 4x master mix. Samples which failed to amplify are denoted as “not detected” (ND). Plasmid DNA was added directly into the master mix at 200,000 copies/mL (2-4x Saliva Direct limit of detection) in triplicate. Full reactions (15μL master mix with 5μL sample input), half reactions (7.5μL master mix with 5μL sample input).

    Techniques Used: Quantitative RT-PCR, Plasmid Preparation

    Limit of detection and reproducibility. a) The limit of detection was defined by extracting RNA from heat-inactivated virus at concentrations ranging from 10.24 to 0.01 TCID 50 /mL and performing RT-qPCR with the LuNER reagents. b) Representative plate controls for the LuNER assay are shown from the LoD titration experiment, including negative buffer-only control (NC), human RNA control (HC), RT-qPCR negative (blank), and RT-qPCR positive controls (synthetic SARS-CoV-2 RNA genome). c) An independent experiment showing reproducibility of the LoD at 0.64, 1.28, and 2.56 TCID 50 /mL.
    Figure Legend Snippet: Limit of detection and reproducibility. a) The limit of detection was defined by extracting RNA from heat-inactivated virus at concentrations ranging from 10.24 to 0.01 TCID 50 /mL and performing RT-qPCR with the LuNER reagents. b) Representative plate controls for the LuNER assay are shown from the LoD titration experiment, including negative buffer-only control (NC), human RNA control (HC), RT-qPCR negative (blank), and RT-qPCR positive controls (synthetic SARS-CoV-2 RNA genome). c) An independent experiment showing reproducibility of the LoD at 0.64, 1.28, and 2.56 TCID 50 /mL.

    Techniques Used: Quantitative RT-PCR, Titration

    15) Product Images from "Modulation of Iron Import and Metronidazole Resistance in Bacteroides fragilis Harboring a nimA Gene"

    Article Title: Modulation of Iron Import and Metronidazole Resistance in Bacteroides fragilis Harboring a nimA Gene

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2022.898453

    RT-qPCR analysis of gene expression in B . fragilis . (A) mRNA levels of hemin import protein HmuY in 638R and 638R nimA before and after induction of metronidazole resistance (64 μg ml −1 ). All strains were measured three times in triplicate. Unpaired t -test (two-tailed): *638R
    Figure Legend Snippet: RT-qPCR analysis of gene expression in B . fragilis . (A) mRNA levels of hemin import protein HmuY in 638R and 638R nimA before and after induction of metronidazole resistance (64 μg ml −1 ). All strains were measured three times in triplicate. Unpaired t -test (two-tailed): *638R

    Techniques Used: Quantitative RT-PCR, Expressing, Two Tailed Test

    16) Product Images from "A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing"

    Article Title: A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing

    Journal: Frontiers in Molecular Biosciences

    doi: 10.3389/fmolb.2022.801309

    A sensitive, robust RT-LAMP assay compatible with crude patient samples. (A) Schematic illustrating loop-mediated amplification (LAMP) of SARS-CoV-2 RNA and the regions targeted in this study (Orf1ab, E and N genes; depicted above). Each target region is recognized by a defined set of primers (B3, LB, BIP, LF, FIP, F3). The RNA template (red) is reverse transcribed and displaced after first-strand synthesis; the outer primer binding sites are added in the subsequent amplification step. The resulting dumbbell DNA structure acts as template for further rounds of amplification, ultimately leading to high molecular weight amplicons. (B) Readout of a real-time fluorescence RT-LAMP reaction using 500 copies of synthetic SARS-CoV-2 (red) or water as non-targeting control (NTC, black) as input. “Time to threshold” indicates the time at which the fluorescence value reaches threshold level (equivalent to Cq value in RT-qPCR assays), “end-point RFU” indicates the fluorescence value (FAM filter set, absorption/emission at 494 nm/518 nm) after 35 min reaction time (used throughout this study unless indicated otherwise); RFU: relative fluorescence units. (C) Performance of the three top primer sets for RT-LAMP-based SARS-CoV-2 detection. End-point relative fluorescence units (RFUs) of RT-LAMP reactions (in duplicates) using the indicated primer sets and serially diluted synthetic SARS-CoV-2 RNA standard as input. Water was used as no-target control (NTC). (D) Cartoon indicating the workflow for SARS-CoV-2 detection by either RT-LAMP or 1-step RT-qPCR from patient samples (nasopharyngeal swab or gargle) with prior RNA isolation. (E) Comparison of RT-LAMP and RT-qPCR performance. Plotted are RT-LAMP end-point fluorescence values after 35 min versus the respective RT-qPCR Cq values. RNA was derived from gargle (green) or nasopharyngeal swabs (black); two no-target controls were included (black cross). Reactions in which no amplification was recorded are labelled as qPCR negative. (F) Detection rate for RT-LAMP reactions compared to a gold standard 1-step RT-qPCR assay. Shown are percentages of positive (detected in RT-LAMP and RT-qPCR) predictive agreement for sample groups (defined by RT-qPCR-derived Cq values) between RT-LAMP (using E- and/or N-gene primers) and 1-step RT-qPCR. (G) Performance of different crude sample preparation methods in RT-LAMP. Shown are end-point relative fluorescence units (RFUs) for RT-LAMP reactions targeting human RNAseP on sample inputs derived from defined numbers of HEK293 cells mixed 1:1 with indicated 2x buffers (extracted RNA served as a positive control). (H) Cartoon indicating the workflow for RT-LAMP using QuickExtract crude lysate as sample input. (I) Comparison of QuickExtract crude sample input versus extracted RNA as input using 1-step RT-qPCR. COVID-19 patient nasopharyngeal swabs or gargle samples (color coded according to the indicated collection medium) were either processed with the QuickExtract workflow (crude sample input) or RNA was extracted using an automated King Fisher RNA bead purification protocol. Reactions in which no amplification was recorded are labelled as qPCR negative. (J) Performance of RT-LAMP with QuickExtract treated crude COVID-19 patient sample input (same samples as in I). Depicted is the comparison of Cq values from RT-qPCR performed on QuickExtract treated samples versus corresponding end-point relative fluorescence units (RFUs) from RT-LAMP reactions.
    Figure Legend Snippet: A sensitive, robust RT-LAMP assay compatible with crude patient samples. (A) Schematic illustrating loop-mediated amplification (LAMP) of SARS-CoV-2 RNA and the regions targeted in this study (Orf1ab, E and N genes; depicted above). Each target region is recognized by a defined set of primers (B3, LB, BIP, LF, FIP, F3). The RNA template (red) is reverse transcribed and displaced after first-strand synthesis; the outer primer binding sites are added in the subsequent amplification step. The resulting dumbbell DNA structure acts as template for further rounds of amplification, ultimately leading to high molecular weight amplicons. (B) Readout of a real-time fluorescence RT-LAMP reaction using 500 copies of synthetic SARS-CoV-2 (red) or water as non-targeting control (NTC, black) as input. “Time to threshold” indicates the time at which the fluorescence value reaches threshold level (equivalent to Cq value in RT-qPCR assays), “end-point RFU” indicates the fluorescence value (FAM filter set, absorption/emission at 494 nm/518 nm) after 35 min reaction time (used throughout this study unless indicated otherwise); RFU: relative fluorescence units. (C) Performance of the three top primer sets for RT-LAMP-based SARS-CoV-2 detection. End-point relative fluorescence units (RFUs) of RT-LAMP reactions (in duplicates) using the indicated primer sets and serially diluted synthetic SARS-CoV-2 RNA standard as input. Water was used as no-target control (NTC). (D) Cartoon indicating the workflow for SARS-CoV-2 detection by either RT-LAMP or 1-step RT-qPCR from patient samples (nasopharyngeal swab or gargle) with prior RNA isolation. (E) Comparison of RT-LAMP and RT-qPCR performance. Plotted are RT-LAMP end-point fluorescence values after 35 min versus the respective RT-qPCR Cq values. RNA was derived from gargle (green) or nasopharyngeal swabs (black); two no-target controls were included (black cross). Reactions in which no amplification was recorded are labelled as qPCR negative. (F) Detection rate for RT-LAMP reactions compared to a gold standard 1-step RT-qPCR assay. Shown are percentages of positive (detected in RT-LAMP and RT-qPCR) predictive agreement for sample groups (defined by RT-qPCR-derived Cq values) between RT-LAMP (using E- and/or N-gene primers) and 1-step RT-qPCR. (G) Performance of different crude sample preparation methods in RT-LAMP. Shown are end-point relative fluorescence units (RFUs) for RT-LAMP reactions targeting human RNAseP on sample inputs derived from defined numbers of HEK293 cells mixed 1:1 with indicated 2x buffers (extracted RNA served as a positive control). (H) Cartoon indicating the workflow for RT-LAMP using QuickExtract crude lysate as sample input. (I) Comparison of QuickExtract crude sample input versus extracted RNA as input using 1-step RT-qPCR. COVID-19 patient nasopharyngeal swabs or gargle samples (color coded according to the indicated collection medium) were either processed with the QuickExtract workflow (crude sample input) or RNA was extracted using an automated King Fisher RNA bead purification protocol. Reactions in which no amplification was recorded are labelled as qPCR negative. (J) Performance of RT-LAMP with QuickExtract treated crude COVID-19 patient sample input (same samples as in I). Depicted is the comparison of Cq values from RT-qPCR performed on QuickExtract treated samples versus corresponding end-point relative fluorescence units (RFUs) from RT-LAMP reactions.

    Techniques Used: RT Lamp Assay, Amplification, Binding Assay, Molecular Weight, Fluorescence, Quantitative RT-PCR, Isolation, Derivative Assay, Real-time Polymerase Chain Reaction, Sample Prep, Positive Control, Purification

    17) Product Images from "A streamlined, cost-effective, and specific method to deplete transcripts for RNA-seq"

    Article Title: A streamlined, cost-effective, and specific method to deplete transcripts for RNA-seq

    Journal: bioRxiv

    doi: 10.1101/2020.05.21.109033

    RNaseH-mediated rRNA and non-specific mRNA depletion. A) Nondenaturing 1.2% agarose gel depicting the following lanes from left to right: 1) ssRNA ladder; 2) total RNA input; 3) input with mock incubations; 4) RNaseH treatment and DNaseI treatment without oligos; 5) DNaseI and oligos only; and 6-8) increasing oligo:RNA mass ratio (1:1, 2:1, and 4:1; total RNA fixed at 1 µg) with RNaseH and DNaseI treatment. B) Fold depletion of 18S rRNA, ACTB, and GAPDH transcripts normalized to input total RNA by RT-qPCR performed on the RNA samples in panel A. Total RNA samples incubated with RNaseH and DNaseI without oligos (grey bar) or increasing oligo:RNA ratio (red bars) with RNaseH and DNaseI treatment. NEB E.coli RNaseH treatment for 1 hour at 37°C and NEB E.coli DNaseI treatment for 30 minutes at 37°C used for samples in this experiment.
    Figure Legend Snippet: RNaseH-mediated rRNA and non-specific mRNA depletion. A) Nondenaturing 1.2% agarose gel depicting the following lanes from left to right: 1) ssRNA ladder; 2) total RNA input; 3) input with mock incubations; 4) RNaseH treatment and DNaseI treatment without oligos; 5) DNaseI and oligos only; and 6-8) increasing oligo:RNA mass ratio (1:1, 2:1, and 4:1; total RNA fixed at 1 µg) with RNaseH and DNaseI treatment. B) Fold depletion of 18S rRNA, ACTB, and GAPDH transcripts normalized to input total RNA by RT-qPCR performed on the RNA samples in panel A. Total RNA samples incubated with RNaseH and DNaseI without oligos (grey bar) or increasing oligo:RNA ratio (red bars) with RNaseH and DNaseI treatment. NEB E.coli RNaseH treatment for 1 hour at 37°C and NEB E.coli DNaseI treatment for 30 minutes at 37°C used for samples in this experiment.

    Techniques Used: Agarose Gel Electrophoresis, Quantitative RT-PCR, Incubation

    18) Product Images from "Exosome-mediated stable epigenetic repression of HIV-1"

    Article Title: Exosome-mediated stable epigenetic repression of HIV-1

    Journal: Nature Communications

    doi: 10.1038/s41467-021-25839-2

    ZPAMt and ZKMt occupy and methylate HIV-1 LTR promoter and repress virus expression long-term manner that is refractory to activation. a RT-qPCR for ZFP mRNA, two days post-transfection in CHI-Ju cells reveals overexpression of mRNA from ZFP362 fusion constructs or pcDNA control (cntrl). b , c ChIP with Myc tag beads reveals ( b ) occupancy of ZFP362 fusion proteins and ZFP362 alone on HIV-1 LTR and ( c ) little to no binding to the non-specific GAPDH region. d RT-qPCR for Gag mRNA, ten days post-transfection of constructs in CHI-Ju cells reveals potent HIV-1 transcription repression from ZFP362 fusion constructs compared to ZFP362 backbone. e , f MeDIP assay from cells transfected with fusion constructs show ( e ) enhancement of CpG methylation on LTR ( f ) no significant changes in CpG methylation on Tat region of NL4-3 genome. For a – f the data represents means ± SD of triplicate treated samples and experiments were repeated twice. p -values were determined by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. **** p ≤ 0.0001. g Treated cultures were assessed by RT-qPCR for GagGag mRNA for two months post-transfection of ZPAMt and ZKMt. h RT-qPCR for Gag mRNA shows a level of reactivation of HIV-1 transcripts from transfected CHI-Ju cells after treatment with various latency reactivation reagents and 0.1% DMSO was used as vehicle control. Each experimental sample was compared to pcDNA transfected control. For g , h data represent mean ± SD of triplicate treated samples. For h p -values were determined by two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. **** p ≤ 0.0001 Source data are provided in the source data file.
    Figure Legend Snippet: ZPAMt and ZKMt occupy and methylate HIV-1 LTR promoter and repress virus expression long-term manner that is refractory to activation. a RT-qPCR for ZFP mRNA, two days post-transfection in CHI-Ju cells reveals overexpression of mRNA from ZFP362 fusion constructs or pcDNA control (cntrl). b , c ChIP with Myc tag beads reveals ( b ) occupancy of ZFP362 fusion proteins and ZFP362 alone on HIV-1 LTR and ( c ) little to no binding to the non-specific GAPDH region. d RT-qPCR for Gag mRNA, ten days post-transfection of constructs in CHI-Ju cells reveals potent HIV-1 transcription repression from ZFP362 fusion constructs compared to ZFP362 backbone. e , f MeDIP assay from cells transfected with fusion constructs show ( e ) enhancement of CpG methylation on LTR ( f ) no significant changes in CpG methylation on Tat region of NL4-3 genome. For a – f the data represents means ± SD of triplicate treated samples and experiments were repeated twice. p -values were determined by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. **** p ≤ 0.0001. g Treated cultures were assessed by RT-qPCR for GagGag mRNA for two months post-transfection of ZPAMt and ZKMt. h RT-qPCR for Gag mRNA shows a level of reactivation of HIV-1 transcripts from transfected CHI-Ju cells after treatment with various latency reactivation reagents and 0.1% DMSO was used as vehicle control. Each experimental sample was compared to pcDNA transfected control. For g , h data represent mean ± SD of triplicate treated samples. For h p -values were determined by two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. **** p ≤ 0.0001 Source data are provided in the source data file.

    Techniques Used: Expressing, Activation Assay, Quantitative RT-PCR, Transfection, Over Expression, Construct, Chromatin Immunoprecipitation, Binding Assay, Methylated DNA Immunoprecipitation, CpG Methylation Assay

    In vivo distribution of exosome cargo and ZPAMt exosome mediated suppression of viral load in HIV-1-infected hu-PBMC-NSG mice. a Schematic illustrating the experimental plan to check the efficacy of ZPAMt exosomes in controlling HIV-1 infection progression in hu-PBMC-NSG mice. b Dynamics of viremia levels in hu-PBMC-NSG mice after treatment with either nLuc exosomes (left), ZFP exosomes (center), ZPAMt exosomes (right) over a course of 10 weeks of infection and treatment as measured by RT-qPCR. The trendline in red indicates the median of the viral load. The pink line indicates the limit of detection (LOD) of the PCR assay is 200 RNA copies/ml in 50–80 μl of mice plasma. c Median values of HIV-1 load monitored periodically in hu-PBMC NSG mice subjected to different treatments i.e. 4 weeks (w) oral cART, 2w oral cART + nLuc or ZFP or ZPAMt exosomes, and no cART mice. Greyline indicates LOD. d MeDIP assay revealed the status of DNA methylation as an effect of treatment of nLuc/ZFP/ZPAMt exosomes on BM-derived cells from HIV-1-infected hu-PBMC-NSG mice. e Toxicity assay was done by measuring Alanine transaminase activity (left) and Aspartate aminotransferase assay activity (right) for all the mice and was compared to activity from untreated NSG mice. Ordinary one-way ANOVA was used to calculate significance P > 0.05 is nonsignificant (ns). In b – e n = 6 was used for nLuc and ZFP treated mice, n = 7 was used for ZPAMt treated mice and in ( c , e ) n = 3 was used for oral cART treated, no cART, hu-PBMC NSG, and untreated NSG. For b , e P value was determined by the Kruskal–Wallis test followed by Dunn’s post hoc test for multiple comparisons. Source data are provided in the source data file.
    Figure Legend Snippet: In vivo distribution of exosome cargo and ZPAMt exosome mediated suppression of viral load in HIV-1-infected hu-PBMC-NSG mice. a Schematic illustrating the experimental plan to check the efficacy of ZPAMt exosomes in controlling HIV-1 infection progression in hu-PBMC-NSG mice. b Dynamics of viremia levels in hu-PBMC-NSG mice after treatment with either nLuc exosomes (left), ZFP exosomes (center), ZPAMt exosomes (right) over a course of 10 weeks of infection and treatment as measured by RT-qPCR. The trendline in red indicates the median of the viral load. The pink line indicates the limit of detection (LOD) of the PCR assay is 200 RNA copies/ml in 50–80 μl of mice plasma. c Median values of HIV-1 load monitored periodically in hu-PBMC NSG mice subjected to different treatments i.e. 4 weeks (w) oral cART, 2w oral cART + nLuc or ZFP or ZPAMt exosomes, and no cART mice. Greyline indicates LOD. d MeDIP assay revealed the status of DNA methylation as an effect of treatment of nLuc/ZFP/ZPAMt exosomes on BM-derived cells from HIV-1-infected hu-PBMC-NSG mice. e Toxicity assay was done by measuring Alanine transaminase activity (left) and Aspartate aminotransferase assay activity (right) for all the mice and was compared to activity from untreated NSG mice. Ordinary one-way ANOVA was used to calculate significance P > 0.05 is nonsignificant (ns). In b – e n = 6 was used for nLuc and ZFP treated mice, n = 7 was used for ZPAMt treated mice and in ( c , e ) n = 3 was used for oral cART treated, no cART, hu-PBMC NSG, and untreated NSG. For b , e P value was determined by the Kruskal–Wallis test followed by Dunn’s post hoc test for multiple comparisons. Source data are provided in the source data file.

    Techniques Used: In Vivo, Infection, Mouse Assay, Quantitative RT-PCR, Polymerase Chain Reaction, Methylated DNA Immunoprecipitation, DNA Methylation Assay, Derivative Assay, Activity Assay

    Exosome mediated delivery of ZPAMt mRNA to virus infected cells. a Schematic for mechanism ZPAMt mRNA transfer from producer cells to recipient cells. HEK293T producer cells are transfected with the following 4 plasmids: “Booster” to increase exosome production, cytosolic delivery helper “Connexin43 S368A”, packaging plasmid CD63-L7Ae, and cargo mRNA-C/D box plasmid. The cargo was intended to be either nLuc reporter, ZFP (non-fusion control), or ZPAMt. Exosomes derived from these producer cells with the ZPAMt-C/D box mRNA can transfer ZPAMt-C/D box mRNA in recipient cells and produce the ZPAMt fusion protein. This ZPAMt protein leads to transcriptional epigenetic repression of HIV-1 within the infected cell. b Concentration and size distribution of exosomes secreted from producer cells transfected with EXOtic system plasmids with either control nLuc-C/D box , ZFP-C/D box , or ZPAMt-C/D box cells engineered with the exosome production booster as measured by Nanoparticle tracking analysis (NTA). TEM image insets show the morphology and size of EXOtic derived exosomes. Scale bars 100 nm. NTA graphs and TEM images representative of n = 3 biological replicates. c Western blot analysis from exosome lysates shown the expression of common exosome protein markers like Alix, CD63, CD81, and Tsg10. Representative blot of n = 2 biological replicates. d Copy number calculation by RT-qPCR for ZFP region reveals packaging of ZFP/ZPAMt mRNA content in exosomes compared to nLuc mRNA packaged exosomes used as a control. e Western blot analysis using an anti-Myc antibody of lysates from ZFP and ZPAMt exosome-treated cells. GAPDH was detected as a loading control. Representative blot of n = 3 biological replicates f RT-qPCR for Gag mRNA was detected in CHI-Ju cells treated with exosomes containing either nLuc (control), ZFP, or ZPAMt. g Gag expression after three doses of HEK-293T derived exosomes on five separate healthy donor-derived, HIV-1-infected PBMCs. PBMCs were treated with exosomes with nLuc loaded RNA to serve as a control. The error bars indicate the standard deviation of triplicate-treated samples. For f , g P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. **** p ≤ 0.0001. Source data are provided in a source data file.
    Figure Legend Snippet: Exosome mediated delivery of ZPAMt mRNA to virus infected cells. a Schematic for mechanism ZPAMt mRNA transfer from producer cells to recipient cells. HEK293T producer cells are transfected with the following 4 plasmids: “Booster” to increase exosome production, cytosolic delivery helper “Connexin43 S368A”, packaging plasmid CD63-L7Ae, and cargo mRNA-C/D box plasmid. The cargo was intended to be either nLuc reporter, ZFP (non-fusion control), or ZPAMt. Exosomes derived from these producer cells with the ZPAMt-C/D box mRNA can transfer ZPAMt-C/D box mRNA in recipient cells and produce the ZPAMt fusion protein. This ZPAMt protein leads to transcriptional epigenetic repression of HIV-1 within the infected cell. b Concentration and size distribution of exosomes secreted from producer cells transfected with EXOtic system plasmids with either control nLuc-C/D box , ZFP-C/D box , or ZPAMt-C/D box cells engineered with the exosome production booster as measured by Nanoparticle tracking analysis (NTA). TEM image insets show the morphology and size of EXOtic derived exosomes. Scale bars 100 nm. NTA graphs and TEM images representative of n = 3 biological replicates. c Western blot analysis from exosome lysates shown the expression of common exosome protein markers like Alix, CD63, CD81, and Tsg10. Representative blot of n = 2 biological replicates. d Copy number calculation by RT-qPCR for ZFP region reveals packaging of ZFP/ZPAMt mRNA content in exosomes compared to nLuc mRNA packaged exosomes used as a control. e Western blot analysis using an anti-Myc antibody of lysates from ZFP and ZPAMt exosome-treated cells. GAPDH was detected as a loading control. Representative blot of n = 3 biological replicates f RT-qPCR for Gag mRNA was detected in CHI-Ju cells treated with exosomes containing either nLuc (control), ZFP, or ZPAMt. g Gag expression after three doses of HEK-293T derived exosomes on five separate healthy donor-derived, HIV-1-infected PBMCs. PBMCs were treated with exosomes with nLuc loaded RNA to serve as a control. The error bars indicate the standard deviation of triplicate-treated samples. For f , g P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. **** p ≤ 0.0001. Source data are provided in a source data file.

    Techniques Used: Infection, Transfection, Plasmid Preparation, Derivative Assay, Concentration Assay, Transmission Electron Microscopy, Western Blot, Expressing, Quantitative RT-PCR, Standard Deviation

    Screening of potent repressors of HIV-1 derived from fusion of ZFP362 and combination of repressor domains. a Schematic representation of the design of the ZD3A constructs leading to ZD3A protein. The NLS containing the ZFP362 domain can bind HIV-1 LTR promoter specifically and DNMT3A can recruit epigenetic silencing complexes. The binding of ZFP362-DNMT3A (ZD3A) can lead to long-term epigenetic transcriptional repression of the LTR represented by methylated CpG DNA and repressive histone methylation (right). b The effect of transfection of ZFP and ZD3A constructs in chronically infected Jurkat (CHI-Ju) cells was measured via RT-qPCR for Gag mRNA at 3 days post-treatment. c Measurement of Gag mRNA from TNFα activated CHI-Ju cells three days after transfection with ZD3A. d The effect of transfection of constructs ZFP and ZD3A in chronically infected Jurkat (CHI-Ju) cells 10 days post-treatment was measured by RT-qPCR for Gag mRNA. e A schematic is shown here depicting the 12 different constructs developed and assessed consisting of ZFP362 with one or more repressor domains like KRAB(K), PWWP, ADD, Methyltransferase (Mtase), Catalytic Domain (CD) of DNMT3A fusions, and the full-length DNMT3a (ZD3A) and control ZFP-362. f CHI-Ju cells were transfected with those fusion constructs depicted in ( e ) and RT-qPCR for Gag mRNA 10 days post-transfection revealed FC4 and FC10 to be the most potent repressors of LTR activity. FC4 will henceforth be referred to as ZPAMt after constituent ZFP362, PWWP, ADD, and Mtase domains, and FC10 will be referred to as KMt after constituent ZFP362, KRAB, and Mtase domains. g Western blot for Myc tag confirmed the expression of ZPAMt, ZKMt, ZD3A, and ZFP. Representative blot of n = 3 biological replicates ( h ) LTR driven GFP mRNA expression level measured after overexpression of ZPAMt and ZKMt in microglial cell line HC69.5 Data are represented as mean ± standard deviation (SD) in b – d , f , and h . p -values by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. For b – d , f , and h the error bars indicate the standard deviation of triplicate treated samples and experiments were repeated twice. **** p ≤ 0.0001. Source data are provided as a source data file.
    Figure Legend Snippet: Screening of potent repressors of HIV-1 derived from fusion of ZFP362 and combination of repressor domains. a Schematic representation of the design of the ZD3A constructs leading to ZD3A protein. The NLS containing the ZFP362 domain can bind HIV-1 LTR promoter specifically and DNMT3A can recruit epigenetic silencing complexes. The binding of ZFP362-DNMT3A (ZD3A) can lead to long-term epigenetic transcriptional repression of the LTR represented by methylated CpG DNA and repressive histone methylation (right). b The effect of transfection of ZFP and ZD3A constructs in chronically infected Jurkat (CHI-Ju) cells was measured via RT-qPCR for Gag mRNA at 3 days post-treatment. c Measurement of Gag mRNA from TNFα activated CHI-Ju cells three days after transfection with ZD3A. d The effect of transfection of constructs ZFP and ZD3A in chronically infected Jurkat (CHI-Ju) cells 10 days post-treatment was measured by RT-qPCR for Gag mRNA. e A schematic is shown here depicting the 12 different constructs developed and assessed consisting of ZFP362 with one or more repressor domains like KRAB(K), PWWP, ADD, Methyltransferase (Mtase), Catalytic Domain (CD) of DNMT3A fusions, and the full-length DNMT3a (ZD3A) and control ZFP-362. f CHI-Ju cells were transfected with those fusion constructs depicted in ( e ) and RT-qPCR for Gag mRNA 10 days post-transfection revealed FC4 and FC10 to be the most potent repressors of LTR activity. FC4 will henceforth be referred to as ZPAMt after constituent ZFP362, PWWP, ADD, and Mtase domains, and FC10 will be referred to as KMt after constituent ZFP362, KRAB, and Mtase domains. g Western blot for Myc tag confirmed the expression of ZPAMt, ZKMt, ZD3A, and ZFP. Representative blot of n = 3 biological replicates ( h ) LTR driven GFP mRNA expression level measured after overexpression of ZPAMt and ZKMt in microglial cell line HC69.5 Data are represented as mean ± standard deviation (SD) in b – d , f , and h . p -values by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. For b – d , f , and h the error bars indicate the standard deviation of triplicate treated samples and experiments were repeated twice. **** p ≤ 0.0001. Source data are provided as a source data file.

    Techniques Used: Derivative Assay, Construct, Binding Assay, Methylation, Transfection, Infection, Quantitative RT-PCR, Activity Assay, Western Blot, Expressing, Over Expression, Standard Deviation

    Delivery of ZPAMt and decline in HIV-1 reservoir in the brain of HIV-1 infected hu-CD34 + NSG mice. a Schematic, illustrating the experimental plan to check the efficacy of ZPAMt exosomes in controlling HIV-1 infection progression in hu-CD34 + NSG mice. b Dynamics of viremia in hu-CD34 + NSG mice after treatment with either nLuc exosomes (left), ZFP exosomes (center), ZPAMt exosomes (right) over the course of 10 weeks of infection and treatment as measured by RT-qPCR. Trendlines in red indicate the median of viral load. The pink line indicates LOD. c Whole-brain obtained after euthanizing HIV-1-infected hu-CD34 + NSG mice were subjected to formalin fixation and were paraffin-embedded. RNA was extracted from 10 micron thick slices from FFPE brain and RT-qPCR for Pol RNA using a Taqman probe was performed to determine the level of HIV-1 Pol mRNA in the brain of nLuc, ZFP, and ZPAMt exosome-treated mice. d , e RT-qPCR for Gag RNA from an equal amount of RNA from ( d ) bone marrow lysate ( e ) spleen tissue lysate from hu-CD34 + NSG mice of nLuc, ZFP, and ZPAMt exosomes treated mice. The line shows the median value in c – e . For b – e n = 7 were used for nLuc treated mice, n = 8 for ZFP treated mice and n = 10 for ZPAMt treated mice. For b , d Kruskal–Wallis test was performed to calculate P value, for c , e P value was determined by Kruskal–Wallis test followed by Dunn’s post hoc test for multiple comparisons. Source data are provided in a source data file.
    Figure Legend Snippet: Delivery of ZPAMt and decline in HIV-1 reservoir in the brain of HIV-1 infected hu-CD34 + NSG mice. a Schematic, illustrating the experimental plan to check the efficacy of ZPAMt exosomes in controlling HIV-1 infection progression in hu-CD34 + NSG mice. b Dynamics of viremia in hu-CD34 + NSG mice after treatment with either nLuc exosomes (left), ZFP exosomes (center), ZPAMt exosomes (right) over the course of 10 weeks of infection and treatment as measured by RT-qPCR. Trendlines in red indicate the median of viral load. The pink line indicates LOD. c Whole-brain obtained after euthanizing HIV-1-infected hu-CD34 + NSG mice were subjected to formalin fixation and were paraffin-embedded. RNA was extracted from 10 micron thick slices from FFPE brain and RT-qPCR for Pol RNA using a Taqman probe was performed to determine the level of HIV-1 Pol mRNA in the brain of nLuc, ZFP, and ZPAMt exosome-treated mice. d , e RT-qPCR for Gag RNA from an equal amount of RNA from ( d ) bone marrow lysate ( e ) spleen tissue lysate from hu-CD34 + NSG mice of nLuc, ZFP, and ZPAMt exosomes treated mice. The line shows the median value in c – e . For b – e n = 7 were used for nLuc treated mice, n = 8 for ZFP treated mice and n = 10 for ZPAMt treated mice. For b , d Kruskal–Wallis test was performed to calculate P value, for c , e P value was determined by Kruskal–Wallis test followed by Dunn’s post hoc test for multiple comparisons. Source data are provided in a source data file.

    Techniques Used: Infection, Mouse Assay, Quantitative RT-PCR, Formalin-fixed Paraffin-Embedded

    19) Product Images from "Mutation of Ebola virus VP35 Ser129 uncouples interferon antagonist and replication functions"

    Article Title: Mutation of Ebola virus VP35 Ser129 uncouples interferon antagonist and replication functions

    Journal: bioRxiv

    doi: 10.1101/726935

    VP35-S129A retains IFN antagonist function. (A) IFN-β promoter activity in the presence of vector control (VC), VP35-WT (WT) or VP35-Ser129Ala (S129A). HEK293T cells were co-transfected with 60 ng of pGL-IFN-β-luc, 10 ng pRL-TK, and decreasing amounts (100, 10, 1.0, 0.1, 0.01 ng) of VC, WT, or S129A plasmid. Twenty-four h after transfection, cells were additionally transfected with IAV-RNA. After 24 h cells were lysed and luciferase activity was measured. (B) RT-qPCR assay of interferon-stimulated gene (ISG) expression upon IAV-RNA stimulation in the presence of VC, WT, or S129A plasmids. HEK293T were transfected with 2500 ng of VC, WT, or S129A plasmid. Twenty-four h after transfection, cells were stimulated with 2500 ng of IAV-RNA. After 24 h, total RNA was extracted, reverse transcribed, and subjected to quantitative real-time PCR (RT-qPCR) for the analysis of ISG15 and 2’-5’-oligoadenylate synthetase (OAS 2’-5’) levels. mRNA expression levels were normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data represent mean ± SD (n=3) of two independent experiments. (C) Effect of VP35-WT and VP35-S129A on ISG56 expression upon poly I:C stimulation. HeLa cells were left untransfected or transfected with VP35-WT or VP35-S129A. Twenty-four h after transfection, cells were left untreated or treated with 10 μg/mL of poly I:C. After 24 h, cells were harvested and subjected to immunoblotting. Data represent mean ± SD. *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001, **** ≤ 0.0001.
    Figure Legend Snippet: VP35-S129A retains IFN antagonist function. (A) IFN-β promoter activity in the presence of vector control (VC), VP35-WT (WT) or VP35-Ser129Ala (S129A). HEK293T cells were co-transfected with 60 ng of pGL-IFN-β-luc, 10 ng pRL-TK, and decreasing amounts (100, 10, 1.0, 0.1, 0.01 ng) of VC, WT, or S129A plasmid. Twenty-four h after transfection, cells were additionally transfected with IAV-RNA. After 24 h cells were lysed and luciferase activity was measured. (B) RT-qPCR assay of interferon-stimulated gene (ISG) expression upon IAV-RNA stimulation in the presence of VC, WT, or S129A plasmids. HEK293T were transfected with 2500 ng of VC, WT, or S129A plasmid. Twenty-four h after transfection, cells were stimulated with 2500 ng of IAV-RNA. After 24 h, total RNA was extracted, reverse transcribed, and subjected to quantitative real-time PCR (RT-qPCR) for the analysis of ISG15 and 2’-5’-oligoadenylate synthetase (OAS 2’-5’) levels. mRNA expression levels were normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data represent mean ± SD (n=3) of two independent experiments. (C) Effect of VP35-WT and VP35-S129A on ISG56 expression upon poly I:C stimulation. HeLa cells were left untransfected or transfected with VP35-WT or VP35-S129A. Twenty-four h after transfection, cells were left untreated or treated with 10 μg/mL of poly I:C. After 24 h, cells were harvested and subjected to immunoblotting. Data represent mean ± SD. *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001, **** ≤ 0.0001.

    Techniques Used: Activity Assay, Plasmid Preparation, Transfection, Luciferase, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction

    20) Product Images from "Long non-coding RNA COL4A2-AS1 facilitates cell proliferation and glycolysis of colorectal cancer cells via miR-20b-5p/hypoxia inducible factor 1 alpha subunit axis"

    Article Title: Long non-coding RNA COL4A2-AS1 facilitates cell proliferation and glycolysis of colorectal cancer cells via miR-20b-5p/hypoxia inducible factor 1 alpha subunit axis

    Journal: Bioengineered

    doi: 10.1080/21655979.2021.1969833

    The expression of COL4A2-AS1 of the colorectal cancer tissues and cell lines were increased. (a): RT-qPCR showed that the mRNA level of COL4A2-AS1 of colorectal cancer tissues was improved. (b): RT-qPCR showed that the mRNA level of COL4A2-AS1 of colorectal cancer cell lines was improved. (c): RT-qPCR showed that the mRNA level of HIF1A of colorectal cancer tissues was improved. D: RT-qPCR showed that the mRNA level of miR-20b-5p of colorectal cancer tissues was decreased. *** P
    Figure Legend Snippet: The expression of COL4A2-AS1 of the colorectal cancer tissues and cell lines were increased. (a): RT-qPCR showed that the mRNA level of COL4A2-AS1 of colorectal cancer tissues was improved. (b): RT-qPCR showed that the mRNA level of COL4A2-AS1 of colorectal cancer cell lines was improved. (c): RT-qPCR showed that the mRNA level of HIF1A of colorectal cancer tissues was improved. D: RT-qPCR showed that the mRNA level of miR-20b-5p of colorectal cancer tissues was decreased. *** P

    Techniques Used: Expressing, Quantitative RT-PCR

    COL4A2-AS1 bound to miR-20b-5p and down-regulated miR-20b-5p expression. (a): LncACTdb predicted the binding region of miR-20b-5p and COL4A2-AS1. The effects of wt-COL4A2-AS1 or mut-COL4A2-AS1 on miR-20b-5p expression were detected by performing dual-luciferase reporter assay on T84 (b) and SW480 cells (c). (d): The expression level of miR-20b-5p was increased by miR-20b-5p mimic. (e–h): RIP experiments were performed on T84 and SW480 cells with Ago2 antibody, and the co-precipitated RNA was subjected to qPCR for COL4A2-AS1 and miR-20b-5p. ** P
    Figure Legend Snippet: COL4A2-AS1 bound to miR-20b-5p and down-regulated miR-20b-5p expression. (a): LncACTdb predicted the binding region of miR-20b-5p and COL4A2-AS1. The effects of wt-COL4A2-AS1 or mut-COL4A2-AS1 on miR-20b-5p expression were detected by performing dual-luciferase reporter assay on T84 (b) and SW480 cells (c). (d): The expression level of miR-20b-5p was increased by miR-20b-5p mimic. (e–h): RIP experiments were performed on T84 and SW480 cells with Ago2 antibody, and the co-precipitated RNA was subjected to qPCR for COL4A2-AS1 and miR-20b-5p. ** P

    Techniques Used: Expressing, Binding Assay, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction

    21) Product Images from "Attenuation of a very virulent Marek's disease herpesvirus (MDV) by codon pair bias deoptimization"

    Article Title: Attenuation of a very virulent Marek's disease herpesvirus (MDV) by codon pair bias deoptimization

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006857

    Quantification of RNA expression and protein production from the recoded UL30 genes. HEK 293T cells were transfected with dual expression plasmids pVITRO2-TagBFP-UL30-EGFP that carried differently recoded UL30 genes fused in frame with EGFP gene. 24 h post transfection RNA expression (A) from the recoded genes was quantified by qPCR, and protein production by flow cytometry (B). The UL30 RNA levels were normalized against the TagBFP levels. We used EGFP fluorescence as a reporter to quantify protein production of the fusion UL30-EGFP genes. The EGFP fluorescence was normalized against the TagBFP fluorescence. P-values were calculated using Kruskal-Wallis H test, * indicates P
    Figure Legend Snippet: Quantification of RNA expression and protein production from the recoded UL30 genes. HEK 293T cells were transfected with dual expression plasmids pVITRO2-TagBFP-UL30-EGFP that carried differently recoded UL30 genes fused in frame with EGFP gene. 24 h post transfection RNA expression (A) from the recoded genes was quantified by qPCR, and protein production by flow cytometry (B). The UL30 RNA levels were normalized against the TagBFP levels. We used EGFP fluorescence as a reporter to quantify protein production of the fusion UL30-EGFP genes. The EGFP fluorescence was normalized against the TagBFP fluorescence. P-values were calculated using Kruskal-Wallis H test, * indicates P

    Techniques Used: RNA Expression, Transfection, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Fluorescence

    Characterization of recoded MDV UL30 mutants. (A) Effect of recoding on UL30 expression from the virus background. CEC were transfected with the parental or mutant BAC clones that carried differently recoded UL30 genes. 24 h post transfection RNA levels of UL29, UL30 and UL42 genes were quantified by qPCR. P-values were calculated using Kruskal-Wallis H test, * indicates P
    Figure Legend Snippet: Characterization of recoded MDV UL30 mutants. (A) Effect of recoding on UL30 expression from the virus background. CEC were transfected with the parental or mutant BAC clones that carried differently recoded UL30 genes. 24 h post transfection RNA levels of UL29, UL30 and UL42 genes were quantified by qPCR. P-values were calculated using Kruskal-Wallis H test, * indicates P

    Techniques Used: Expressing, Capillary Electrochromatography, Transfection, Mutagenesis, BAC Assay, Clone Assay, Real-time Polymerase Chain Reaction

    22) Product Images from "How Localized Z-Disc Damage Affects Force Generation and Gene Expression in Cardiomyocytes"

    Article Title: How Localized Z-Disc Damage Affects Force Generation and Gene Expression in Cardiomyocytes

    Journal: Bioengineering

    doi: 10.3390/bioengineering8120213

    RT-qPCR analysis of injury and contraction related gene expression comparing control CMs and CMs, which were laser manipulated ( n ≥ 3 experiments). TNNI3 and TNNT2 were significantly (*) downregulated. All data were calculated according to Taylor et al. [ 39 ] and are normalized to the mean of the control group, small red dots present raw data.
    Figure Legend Snippet: RT-qPCR analysis of injury and contraction related gene expression comparing control CMs and CMs, which were laser manipulated ( n ≥ 3 experiments). TNNI3 and TNNT2 were significantly (*) downregulated. All data were calculated according to Taylor et al. [ 39 ] and are normalized to the mean of the control group, small red dots present raw data.

    Techniques Used: Quantitative RT-PCR, Expressing

    RT-qPCR analysis of stress-related gene expression comparing control CMs and CMs, which were laser manipulated ( n ≥ 3 experiments). NF-kB was significantly upregulated (***) after Z-disc removal. All data were calculated according to Taylor et al. [ 39 ] and are normalized to the mean of the control group, small red dots present raw data.
    Figure Legend Snippet: RT-qPCR analysis of stress-related gene expression comparing control CMs and CMs, which were laser manipulated ( n ≥ 3 experiments). NF-kB was significantly upregulated (***) after Z-disc removal. All data were calculated according to Taylor et al. [ 39 ] and are normalized to the mean of the control group, small red dots present raw data.

    Techniques Used: Quantitative RT-PCR, Expressing

    RT-qPCR analysis of sarcomeric cytoskeleton related gene expression comparing control CMs and CMs, which were laser manipulated ( n ≥ 3 experiments). ACTN2 and FLNc were significantly (ACTN: *, FLNC: **) upregulated. All data were calculated according to Taylor et al. [ 39 ] and are normalized to the mean of the control group, small red dots present raw data.
    Figure Legend Snippet: RT-qPCR analysis of sarcomeric cytoskeleton related gene expression comparing control CMs and CMs, which were laser manipulated ( n ≥ 3 experiments). ACTN2 and FLNc were significantly (ACTN: *, FLNC: **) upregulated. All data were calculated according to Taylor et al. [ 39 ] and are normalized to the mean of the control group, small red dots present raw data.

    Techniques Used: Quantitative RT-PCR, Expressing

    23) Product Images from "Identification of microRNA-mRNA regulatory network associated with oxidative DNA damage in human astrocytes"

    Article Title: Identification of microRNA-mRNA regulatory network associated with oxidative DNA damage in human astrocytes

    Journal: ASN NEURO

    doi: 10.1177/17590914221101704

    RT-qPCR confirmed (A) downregulation of miR-335, and (B) upregulation of its target mRNA, PARP-1 (p
    Figure Legend Snippet: RT-qPCR confirmed (A) downregulation of miR-335, and (B) upregulation of its target mRNA, PARP-1 (p

    Techniques Used: Quantitative RT-PCR

    Sodium dichromate increases oxidative DNA base damage. The alkaline comet assay with FPG treatment was used to detect oxidative base damage following 10 μM Na 2 Cr 2 O 7 treatment for 16 h (A) and the tail moment was measured using OpenComet (B). Analysis was performed on one experiment with at least 70 cells in each experimental group. Error bars represent SD and **** represents P
    Figure Legend Snippet: Sodium dichromate increases oxidative DNA base damage. The alkaline comet assay with FPG treatment was used to detect oxidative base damage following 10 μM Na 2 Cr 2 O 7 treatment for 16 h (A) and the tail moment was measured using OpenComet (B). Analysis was performed on one experiment with at least 70 cells in each experimental group. Error bars represent SD and **** represents P

    Techniques Used: Alkaline Single Cell Gel Electrophoresis

    24) Product Images from "Synergism of interferon-beta with antiviral drugs against SARS-CoV-2 variants"

    Article Title: Synergism of interferon-beta with antiviral drugs against SARS-CoV-2 variants

    Journal: bioRxiv

    doi: 10.1101/2022.07.22.501169

    Replication kinetics of SARS-CoV-2 Delta, Omicron BA.1, and Omicron BA.2 isolates in Caco-2 cells. A) Representative immunofluorescence images indicating the number of Spike (S) protein-positive Caco-2-F03 cells 24h and 48h post infection with Delta, BA.1, and BA.2 at an MOI of 1. B) Quantification of S protein-positive Caco-2-F03 cells 24h and 48h post infection with Delta, BA.1, and BA.2 at an MOI of 1. C) Genomic RNA copy numbers determined by qPCR 24h and 48h post infection of Caco-2 cells with Delta, BA.1, and BA.2 at an MOI of 1. D) Cell viability in Caco-2-F03 cells 24h and 48h post infection as determined by CellTiter-Glo ® Luminescent Cell Viability Assay (Promega). Values represent mean ± S.D. of three independent experiments. P-values represent statistical differences between Delta and BA.1 or BA.2 calculated by one-way ANOVA and Tukey’s test. These differences in the replication kinetics (Delta > BA.2 > BA.1) were also reflected in cytopathogenic effect (CPE) formation ( Figure 2A ) and cell viability measurements ( Figure 2D ).
    Figure Legend Snippet: Replication kinetics of SARS-CoV-2 Delta, Omicron BA.1, and Omicron BA.2 isolates in Caco-2 cells. A) Representative immunofluorescence images indicating the number of Spike (S) protein-positive Caco-2-F03 cells 24h and 48h post infection with Delta, BA.1, and BA.2 at an MOI of 1. B) Quantification of S protein-positive Caco-2-F03 cells 24h and 48h post infection with Delta, BA.1, and BA.2 at an MOI of 1. C) Genomic RNA copy numbers determined by qPCR 24h and 48h post infection of Caco-2 cells with Delta, BA.1, and BA.2 at an MOI of 1. D) Cell viability in Caco-2-F03 cells 24h and 48h post infection as determined by CellTiter-Glo ® Luminescent Cell Viability Assay (Promega). Values represent mean ± S.D. of three independent experiments. P-values represent statistical differences between Delta and BA.1 or BA.2 calculated by one-way ANOVA and Tukey’s test. These differences in the replication kinetics (Delta > BA.2 > BA.1) were also reflected in cytopathogenic effect (CPE) formation ( Figure 2A ) and cell viability measurements ( Figure 2D ).

    Techniques Used: Immunofluorescence, Infection, Real-time Polymerase Chain Reaction, Cell Viability Assay

    25) Product Images from "eDNA-stimulated cell dispersion from Caulobacter crescentus biofilms upon oxygen limitation is dependent on a toxin-antitoxin system"

    Article Title: eDNA-stimulated cell dispersion from Caulobacter crescentus biofilms upon oxygen limitation is dependent on a toxin-antitoxin system

    Journal: bioRxiv

    doi: 10.1101/2022.06.17.496608

    eDNA release and biofilm formation under variable O 2 availability. (A) Images of cultures providing different amounts of O 2 , termed maximal, high, moderate, and limited aeration, respectively. (B) Assessment of P parDE4 and P ccoN expression (white and blue bars respectively) by measuring ß-galactosidase activity of the P parDE4 - lacZ transcriptional fusions in WT, and transcription of ccoN relative to rpoD as a function of various aeration conditions by RT-qPCR. (C) Ratio of the eDNA release concentration measured in Δ parDE 4 planktonic phase over WT, in cultures grown under the different aeration conditions. Results are given as the calculated ratio of the eDNA concentration measured in 6 independent replicates, each run in duplicate, and error bars represent the SEM. Statistical comparisons to “maximal aeration” conditions are calculated using Student’s unpaired t-tests;* P
    Figure Legend Snippet: eDNA release and biofilm formation under variable O 2 availability. (A) Images of cultures providing different amounts of O 2 , termed maximal, high, moderate, and limited aeration, respectively. (B) Assessment of P parDE4 and P ccoN expression (white and blue bars respectively) by measuring ß-galactosidase activity of the P parDE4 - lacZ transcriptional fusions in WT, and transcription of ccoN relative to rpoD as a function of various aeration conditions by RT-qPCR. (C) Ratio of the eDNA release concentration measured in Δ parDE 4 planktonic phase over WT, in cultures grown under the different aeration conditions. Results are given as the calculated ratio of the eDNA concentration measured in 6 independent replicates, each run in duplicate, and error bars represent the SEM. Statistical comparisons to “maximal aeration” conditions are calculated using Student’s unpaired t-tests;* P

    Techniques Used: Expressing, Activity Assay, Quantitative RT-PCR, Concentration Assay

    26) Product Images from "Attenuation of a very virulent Marek's disease herpesvirus (MDV) by codon pair bias deoptimization"

    Article Title: Attenuation of a very virulent Marek's disease herpesvirus (MDV) by codon pair bias deoptimization

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006857

    Quantification of RNA expression and protein production from the recoded UL30 genes. HEK 293T cells were transfected with dual expression plasmids pVITRO2-TagBFP-UL30-EGFP that carried differently recoded UL30 genes fused in frame with EGFP gene. 24 h post transfection RNA expression (A) from the recoded genes was quantified by qPCR, and protein production by flow cytometry (B). The UL30 RNA levels were normalized against the TagBFP levels. We used EGFP fluorescence as a reporter to quantify protein production of the fusion UL30-EGFP genes. The EGFP fluorescence was normalized against the TagBFP fluorescence. P-values were calculated using Kruskal-Wallis H test, * indicates P
    Figure Legend Snippet: Quantification of RNA expression and protein production from the recoded UL30 genes. HEK 293T cells were transfected with dual expression plasmids pVITRO2-TagBFP-UL30-EGFP that carried differently recoded UL30 genes fused in frame with EGFP gene. 24 h post transfection RNA expression (A) from the recoded genes was quantified by qPCR, and protein production by flow cytometry (B). The UL30 RNA levels were normalized against the TagBFP levels. We used EGFP fluorescence as a reporter to quantify protein production of the fusion UL30-EGFP genes. The EGFP fluorescence was normalized against the TagBFP fluorescence. P-values were calculated using Kruskal-Wallis H test, * indicates P

    Techniques Used: RNA Expression, Transfection, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Fluorescence

    Characterization of recoded MDV UL30 mutants. (A) Effect of recoding on UL30 expression from the virus background. CEC were transfected with the parental or mutant BAC clones that carried differently recoded UL30 genes. 24 h post transfection RNA levels of UL29, UL30 and UL42 genes were quantified by qPCR. P-values were calculated using Kruskal-Wallis H test, * indicates P
    Figure Legend Snippet: Characterization of recoded MDV UL30 mutants. (A) Effect of recoding on UL30 expression from the virus background. CEC were transfected with the parental or mutant BAC clones that carried differently recoded UL30 genes. 24 h post transfection RNA levels of UL29, UL30 and UL42 genes were quantified by qPCR. P-values were calculated using Kruskal-Wallis H test, * indicates P

    Techniques Used: Expressing, Capillary Electrochromatography, Transfection, Mutagenesis, BAC Assay, Clone Assay, Real-time Polymerase Chain Reaction

    27) Product Images from "Human POT1 Prevents Severe Telomere Instability Induced by Homology Directed DNA Repair"

    Article Title: Human POT1 Prevents Severe Telomere Instability Induced by Homology Directed DNA Repair

    Journal: bioRxiv

    doi: 10.1101/2020.01.20.912642

    (A) Telomere repeat amplification protocol (TRAP) assay for clone 35 treated with 0.5 µM 4OHT and/or 1µM GRN163L. As negative controls, EDTA-containing extracts were run in parallel. (B) Western blot for POT1, DDR markers and recombination proteins in clone 35 upon depletion of selected recombination proteins with siRNA and removal of POT1. (C) qPCR to confirm depletion of BRCA1 and BARD1 in clone 35 upon depletion of these proteins with siRNA. (D) Western blot for POT1, γH2AX BLM in clone 35 upon depletion of BLM with siRNA and removal of POT1. All experiments were performed in triplicate.
    Figure Legend Snippet: (A) Telomere repeat amplification protocol (TRAP) assay for clone 35 treated with 0.5 µM 4OHT and/or 1µM GRN163L. As negative controls, EDTA-containing extracts were run in parallel. (B) Western blot for POT1, DDR markers and recombination proteins in clone 35 upon depletion of selected recombination proteins with siRNA and removal of POT1. (C) qPCR to confirm depletion of BRCA1 and BARD1 in clone 35 upon depletion of these proteins with siRNA. (D) Western blot for POT1, γH2AX BLM in clone 35 upon depletion of BLM with siRNA and removal of POT1. All experiments were performed in triplicate.

    Techniques Used: Amplification, TRAP Assay, Western Blot, Real-time Polymerase Chain Reaction

    28) Product Images from "The Thiazole-5-Carboxamide GPS491 Inhibits HIV-1, Adenovirus, and Coronavirus Replication by Altering RNA Processing/Accumulation"

    Article Title: The Thiazole-5-Carboxamide GPS491 Inhibits HIV-1, Adenovirus, and Coronavirus Replication by Altering RNA Processing/Accumulation

    Journal: Viruses

    doi: 10.3390/v14010060

    GPS491 inhibits replication of 229E, OC43, and SARS-CoV2 coronaviruses. ( a ) Huh7 cells were infected with either 229E or OC43 at an input MOI of 0.1 and 1, respectively, for 1 h. Virus inoculum was removed, cells were washed, and fresh media containing 1% DMSO or varying concentrations of GPS491 (from 0 µM to 10 µM) were added. Then, 2 days p.i (229E) or 4 days p.i. (OC43), media were harvested to quantitate viral genomic RNA levels by RT-qPCR assay. All the values are expressed relative to the values detected in virus-infected DMSO-treated samples. Effect of GPS491 on cell viability was assessed 2 days post compound addition using alamarBlue at the indicated doses of GPS491. Data are indicated as mean ± SD, ** p ≤ 0.01, and *** p ≤ 0.001. ( b , c ) Huh7 cells were infected with ( b ) 229E at an MOI of 0.03 or ( c ) OC43 at an MOI of 0.3 for 1 h after which the virus inoculum was removed, cells were washed with 1× PBS, and media containing 1% DMSO or 1.25 µM GPS491 were added. Cells and media were harvested 2 days (229E) or 4 days (OC43) p.i., and the levels of viral proteins (N, S) were determined by Western blot and virus production was assessed by RT-qPCR of the media (RNA). Shown are representative Western blots and their respective quantitation indicating the effects of GPS491 following infection with 229E and OC43, respectively. Band intensity was quantified relative to virus-infected control and normalized to total protein load (stain-free gels). Data are indicated as mean ± SD and generated from n = 3 independent assays, each performed in duplicate. ( d , e ) Huh7 cells were infected with SARS-CoV2 at an MOI of 1 for 1 h. Virus inoculum was removed, and fresh media containing DMSO, chloroquine (CQ, 10 µM), or indicated doses of GPS491 were added. Two days post infection, ( d ) media were harvested, and levels of virion production determined by RT-qPCR or ( e ) lysates from cells treated with 1% DMSO or 0.3 µM GPS491 were blotted to measure expression of viral N protein. Results shown were generated from n = 3 independent assays.
    Figure Legend Snippet: GPS491 inhibits replication of 229E, OC43, and SARS-CoV2 coronaviruses. ( a ) Huh7 cells were infected with either 229E or OC43 at an input MOI of 0.1 and 1, respectively, for 1 h. Virus inoculum was removed, cells were washed, and fresh media containing 1% DMSO or varying concentrations of GPS491 (from 0 µM to 10 µM) were added. Then, 2 days p.i (229E) or 4 days p.i. (OC43), media were harvested to quantitate viral genomic RNA levels by RT-qPCR assay. All the values are expressed relative to the values detected in virus-infected DMSO-treated samples. Effect of GPS491 on cell viability was assessed 2 days post compound addition using alamarBlue at the indicated doses of GPS491. Data are indicated as mean ± SD, ** p ≤ 0.01, and *** p ≤ 0.001. ( b , c ) Huh7 cells were infected with ( b ) 229E at an MOI of 0.03 or ( c ) OC43 at an MOI of 0.3 for 1 h after which the virus inoculum was removed, cells were washed with 1× PBS, and media containing 1% DMSO or 1.25 µM GPS491 were added. Cells and media were harvested 2 days (229E) or 4 days (OC43) p.i., and the levels of viral proteins (N, S) were determined by Western blot and virus production was assessed by RT-qPCR of the media (RNA). Shown are representative Western blots and their respective quantitation indicating the effects of GPS491 following infection with 229E and OC43, respectively. Band intensity was quantified relative to virus-infected control and normalized to total protein load (stain-free gels). Data are indicated as mean ± SD and generated from n = 3 independent assays, each performed in duplicate. ( d , e ) Huh7 cells were infected with SARS-CoV2 at an MOI of 1 for 1 h. Virus inoculum was removed, and fresh media containing DMSO, chloroquine (CQ, 10 µM), or indicated doses of GPS491 were added. Two days post infection, ( d ) media were harvested, and levels of virion production determined by RT-qPCR or ( e ) lysates from cells treated with 1% DMSO or 0.3 µM GPS491 were blotted to measure expression of viral N protein. Results shown were generated from n = 3 independent assays.

    Techniques Used: Infection, Quantitative RT-PCR, Western Blot, Quantitation Assay, Staining, Generated, Expressing

    GPS491 alters adenovirus RNA expression/processing and inhibits adenovirus DNA amplification. A549 cells were infected with HAdV-C5 at an input MOI of 100 IU/cell for 1 h after which virus inoculum was removed and replaced with media containing DMSO or GPS491 (2.5 or 5 µM). ( a ) Total RNA was extracted at 8 h, 16 h, or 24 h after virus infection. After cDNA generation, RT-qPCR was performed using primers for E1A, E1B, E2A, E2B, and E4 RNAs ( Supplementary Table S1 ). Values are expressed relative to RNA abundance observed 24 h p.i. in the presence of 1% DMSO. Shown are data from samples treated with 2.5 µM GPS491. ( b ) The effect of GPS491 on E1A RNA processing. Shown on the top is the schematic diagram of E1A RNA processing indicating the major E1A mRNA isoforms generated by alternative splicing. In the middle is a representative gel of the E1A RNA amplicons generated from cDNA. The graph on the bottom represents quantitation of amplicons generated across n > 3 independent assays. ( c ) At 16 h, 20 h, and 24 h p.i., total DNA was isolated from cells treated with DMSO or 2.5 µM GPS491 and levels of adenoviral DNA were determined by qPCR. Data are indicated as mean ± SD, * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.
    Figure Legend Snippet: GPS491 alters adenovirus RNA expression/processing and inhibits adenovirus DNA amplification. A549 cells were infected with HAdV-C5 at an input MOI of 100 IU/cell for 1 h after which virus inoculum was removed and replaced with media containing DMSO or GPS491 (2.5 or 5 µM). ( a ) Total RNA was extracted at 8 h, 16 h, or 24 h after virus infection. After cDNA generation, RT-qPCR was performed using primers for E1A, E1B, E2A, E2B, and E4 RNAs ( Supplementary Table S1 ). Values are expressed relative to RNA abundance observed 24 h p.i. in the presence of 1% DMSO. Shown are data from samples treated with 2.5 µM GPS491. ( b ) The effect of GPS491 on E1A RNA processing. Shown on the top is the schematic diagram of E1A RNA processing indicating the major E1A mRNA isoforms generated by alternative splicing. In the middle is a representative gel of the E1A RNA amplicons generated from cDNA. The graph on the bottom represents quantitation of amplicons generated across n > 3 independent assays. ( c ) At 16 h, 20 h, and 24 h p.i., total DNA was isolated from cells treated with DMSO or 2.5 µM GPS491 and levels of adenoviral DNA were determined by qPCR. Data are indicated as mean ± SD, * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.

    Techniques Used: RNA Expression, Amplification, Infection, Quantitative RT-PCR, Generated, Quantitation Assay, Isolation, Real-time Polymerase Chain Reaction

    Effects of GPS491 on HIV-1 RNA accumulation and processing. HeLa rtTA HIV∆mls cells were treated with DMSO or varying concentrations of GPS491 +/− doxycycline (Dox) for 24 h. Cells were then harvested, total RNA was extracted, and cDNA was generated. Samples were subsequently analyzed ( a ) by RT-qPCR to measure levels of HIV-1 US, SS, and MS RNA levels or ( b ) by RT-PCR to measure levels of different MS RNA isoforms. Shown is a representative gel and summary quantitation of results from n > 3 independent assays. Data are indicated as mean ± SD, * p ≤ 0.05, ** p ≤ 0.01, ns—not significant.
    Figure Legend Snippet: Effects of GPS491 on HIV-1 RNA accumulation and processing. HeLa rtTA HIV∆mls cells were treated with DMSO or varying concentrations of GPS491 +/− doxycycline (Dox) for 24 h. Cells were then harvested, total RNA was extracted, and cDNA was generated. Samples were subsequently analyzed ( a ) by RT-qPCR to measure levels of HIV-1 US, SS, and MS RNA levels or ( b ) by RT-PCR to measure levels of different MS RNA isoforms. Shown is a representative gel and summary quantitation of results from n > 3 independent assays. Data are indicated as mean ± SD, * p ≤ 0.05, ** p ≤ 0.01, ns—not significant.

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

    29) Product Images from "A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics"

    Article Title: A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics

    Journal: Nature Communications

    doi: 10.1038/s41467-020-18130-3

    SARS-CoV-2 platform validation using patient samples. a Schematic of a typical workflow tracking viral copy number from a hypothetical patient sample where an input of 200 µL with a minimum 1 particle/µL results in 3–4 copies of extracted RNA per microliter resulting in a range of 15–20 RNA copies per qPCR reaction. b Comparison of RT-qPCR Ct value results for the same 173 patient samples obtained by the North West London Pathology (NWLP) laboratory and the London Biofoundry (LBF), based on the VLP workflow using the Analytik Jena RNA extraction kit and CDC N1 primer–probe set. c Validation of the Promega RNA extraction kit using 65 patient samples with the Analytik Jena RNA extraction workflow previously validated in b . Error bands in b and c represent the 95% confidence interval. d Validation of the CRISPR NAT using patient samples with Ct values determined via RT-qPCR. Error bars represent the mean ± SE of n = 3 independent amplification replicates and four technical replicates for CRISPR detection. e Validation of the RT-LAMP colorimetric NAT using patient samples with Ct values determined via RT-qPCR. Error bars represent the mean ± SE of n = 3 independent amplification reactions. Source data are available in the Source Data file.
    Figure Legend Snippet: SARS-CoV-2 platform validation using patient samples. a Schematic of a typical workflow tracking viral copy number from a hypothetical patient sample where an input of 200 µL with a minimum 1 particle/µL results in 3–4 copies of extracted RNA per microliter resulting in a range of 15–20 RNA copies per qPCR reaction. b Comparison of RT-qPCR Ct value results for the same 173 patient samples obtained by the North West London Pathology (NWLP) laboratory and the London Biofoundry (LBF), based on the VLP workflow using the Analytik Jena RNA extraction kit and CDC N1 primer–probe set. c Validation of the Promega RNA extraction kit using 65 patient samples with the Analytik Jena RNA extraction workflow previously validated in b . Error bands in b and c represent the 95% confidence interval. d Validation of the CRISPR NAT using patient samples with Ct values determined via RT-qPCR. Error bars represent the mean ± SE of n = 3 independent amplification replicates and four technical replicates for CRISPR detection. e Validation of the RT-LAMP colorimetric NAT using patient samples with Ct values determined via RT-qPCR. Error bars represent the mean ± SE of n = 3 independent amplification reactions. Source data are available in the Source Data file.

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, RNA Extraction, CRISPR, Amplification

    MS2-SARS-CoV-2 VLP detection with multiple target primer–probe sets and qPCR master mixes. a VLP dilutions of 2.5, 25, and 250 copies per reaction were analysed by one-step RT-qPCR using the CDC primer–probe sets N1, N2, and N3 with the TaqPath master mix (Thermo Fisher Scientific) and reported as Ct values. b A Ct value standard curve for VLP concentrations of 1, 5, 10, 15, 20, 25, 50, 100, 150, 200, and 250 VLP copies per reaction was determined using the N1 primer–probe set and the TaqPath master mix. c VLP dilutions of 250, 25, and 2.5 copies per reaction were analysed using the TaqPath, Luna Universal (NEB), and Fast Virus (Thermo Fisher Scientific) RT-qPCR master mixes with the N1, N2, and N3 CDC primer–probe sets. All measurements in a and c are reported as mean ± SE of n = 3 independent experiments with three technical replicates. Measurements in b are reported as mean ± SD and are representative of n = 2 independent experiments with three technical replicates. Statistical difference between the TaqPath and Luna, as well as TaqPath and Fast Virus master mix Ct values was analysed using an unpaired two-sided t -test with (black star) indicating p
    Figure Legend Snippet: MS2-SARS-CoV-2 VLP detection with multiple target primer–probe sets and qPCR master mixes. a VLP dilutions of 2.5, 25, and 250 copies per reaction were analysed by one-step RT-qPCR using the CDC primer–probe sets N1, N2, and N3 with the TaqPath master mix (Thermo Fisher Scientific) and reported as Ct values. b A Ct value standard curve for VLP concentrations of 1, 5, 10, 15, 20, 25, 50, 100, 150, 200, and 250 VLP copies per reaction was determined using the N1 primer–probe set and the TaqPath master mix. c VLP dilutions of 250, 25, and 2.5 copies per reaction were analysed using the TaqPath, Luna Universal (NEB), and Fast Virus (Thermo Fisher Scientific) RT-qPCR master mixes with the N1, N2, and N3 CDC primer–probe sets. All measurements in a and c are reported as mean ± SE of n = 3 independent experiments with three technical replicates. Measurements in b are reported as mean ± SD and are representative of n = 2 independent experiments with three technical replicates. Statistical difference between the TaqPath and Luna, as well as TaqPath and Fast Virus master mix Ct values was analysed using an unpaired two-sided t -test with (black star) indicating p

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

    SARS-CoV-2 platform development using the MS2-SARS-CoV-2 VLP standard. a Schematic of the modular platform for the detection of SARS-CoV-2. Viral RNA is isolated using the Analytik Jena FeliX liquid handler with the Analytik Jena or Promega RNA extraction kits. Sample RNA is transferred and detection reactions are set up using the Labcyte Echo platform. These include qPCR, validated for the TaqPath (Thermo Fisher Scientific), Luna (NEB), and Fast Virus (Thermo Fisher Scientific) RT-qPCR master mix options, RT-LAMP nucleic acid detection, and the CRISPR-Cas13a diagnostic workflow. b Automated RNA extraction was developed using VLP dilutions of 10 3 and 10 4 copies/mL for the Analytik Jena and Promega RNA extraction kits. Efficiency of the extractions using both kits was analysed by RT-qPCR with the CDC N1 primer–probe set using the TaqPath master mix. c Dilutions of VLPs used for RT-qPCR in b were analysed using the CRISPR NAT to demonstrate the use of this workflow as an alternative diagnostic option. Error bars in b represent mean ± SE of n = 3 biologically independent samples with three technical replicates. Error bars in c represent mean ± SE of n = 3 independent amplification replicates and four technical replicates for CRISPR detection. Source data are available in the Source Data file.
    Figure Legend Snippet: SARS-CoV-2 platform development using the MS2-SARS-CoV-2 VLP standard. a Schematic of the modular platform for the detection of SARS-CoV-2. Viral RNA is isolated using the Analytik Jena FeliX liquid handler with the Analytik Jena or Promega RNA extraction kits. Sample RNA is transferred and detection reactions are set up using the Labcyte Echo platform. These include qPCR, validated for the TaqPath (Thermo Fisher Scientific), Luna (NEB), and Fast Virus (Thermo Fisher Scientific) RT-qPCR master mix options, RT-LAMP nucleic acid detection, and the CRISPR-Cas13a diagnostic workflow. b Automated RNA extraction was developed using VLP dilutions of 10 3 and 10 4 copies/mL for the Analytik Jena and Promega RNA extraction kits. Efficiency of the extractions using both kits was analysed by RT-qPCR with the CDC N1 primer–probe set using the TaqPath master mix. c Dilutions of VLPs used for RT-qPCR in b were analysed using the CRISPR NAT to demonstrate the use of this workflow as an alternative diagnostic option. Error bars in b represent mean ± SE of n = 3 biologically independent samples with three technical replicates. Error bars in c represent mean ± SE of n = 3 independent amplification replicates and four technical replicates for CRISPR detection. Source data are available in the Source Data file.

    Techniques Used: Isolation, RNA Extraction, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CRISPR, Diagnostic Assay, Amplification

    30) Product Images from "Signal Recognition Particle RNA Contributes to Oxidative Stress Response in Deinococcus radiodurans by Modulating Catalase Localization"

    Article Title: Signal Recognition Particle RNA Contributes to Oxidative Stress Response in Deinococcus radiodurans by Modulating Catalase Localization

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.613571

    Knockdown (KD) of Qpr6 leads to the hypersensitivity of D. radiodurans to H 2 O 2 and chronic ionizing radiation (CIR). (A) Evaluation of Qpr6 level in R1, Qpr6 KD, and Qpr6 Com by RT-qPCR; (B) Survival of R1, Qpr6 KD, and Qpr6 Com under H 2 O 2 stress; (C) Growth of R1, Qpr6 KD, and Qpr6 Com under 0 and 57 Gy/h radiation ( γ -ray) for 6 days; (D) Survival rate of R1, Qpr6 KD, and Qpr6 Com under 10 kGy ionizing radiation ( β -ray). Error bars indicate standard errors of the means ( n = 3). ** p ≤ 0.01, and *** p ≤ 0.001 were considered as significant using the Student t -test, while ns indicates non-significant compared to R1 ( p > 0.05).
    Figure Legend Snippet: Knockdown (KD) of Qpr6 leads to the hypersensitivity of D. radiodurans to H 2 O 2 and chronic ionizing radiation (CIR). (A) Evaluation of Qpr6 level in R1, Qpr6 KD, and Qpr6 Com by RT-qPCR; (B) Survival of R1, Qpr6 KD, and Qpr6 Com under H 2 O 2 stress; (C) Growth of R1, Qpr6 KD, and Qpr6 Com under 0 and 57 Gy/h radiation ( γ -ray) for 6 days; (D) Survival rate of R1, Qpr6 KD, and Qpr6 Com under 10 kGy ionizing radiation ( β -ray). Error bars indicate standard errors of the means ( n = 3). ** p ≤ 0.01, and *** p ≤ 0.001 were considered as significant using the Student t -test, while ns indicates non-significant compared to R1 ( p > 0.05).

    Techniques Used: Quantitative RT-PCR

    31) Product Images from "SARS-CoV-2 Employ BSG/CD147 and ACE2 Receptors to Directly Infect Human Induced Pluripotent Stem Cell-Derived Kidney Podocytes"

    Article Title: SARS-CoV-2 Employ BSG/CD147 and ACE2 Receptors to Directly Infect Human Induced Pluripotent Stem Cell-Derived Kidney Podocytes

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2022.855340

    Susceptibility of human iPS cell-derived podocytes to infection by live SARS-CoV-2. qPCR analysis of human iPS cell-derived podocytes infected with SARS-CoV-2 revealed intracellular uptake of the virus for 24 h.p.i (A) , 48 h.p.i (B) and 72 h.p.i (C) . (D) plaque assay quantification from supernatant obtained from infected podocytes at 24, 48 and 72 h.p.i. (E) qPCR analysis of podocyte-specific genes revealed that both synaptopodin (SYNPO) and podocalyxin (PODXL) are significantly upregulated after infection with SARS-CoV-2 at MOI of 0.01, whereas SYNPO is significantly upregulated at multiple MOIs, and PODXL shows no significant changes with viral infection. (F) The expression of spike-associated genes (ACE2, BSG/CD147) and spike processing genes (TMPRSS2, CTSL) are significantly impacted by infection at MOIs of 0.01 and 0.1, respectively. (G) Human iPS cell-derived podocytes treated with SARS-CoV-2 (at MOI of 0.01) immunostain positive for Nucleocapsid protein (magenta), indicating successful infection with the virus. The cells were immunostained also for the podocyte marker Nephrin (green) and counterstained with DAPI (blue). Scale bar: 100 µm (H) Spike positive cells Nephrin and DAPI as nuclear counterstain in the infected podocytes. Scale bar: 100 µm. One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p
    Figure Legend Snippet: Susceptibility of human iPS cell-derived podocytes to infection by live SARS-CoV-2. qPCR analysis of human iPS cell-derived podocytes infected with SARS-CoV-2 revealed intracellular uptake of the virus for 24 h.p.i (A) , 48 h.p.i (B) and 72 h.p.i (C) . (D) plaque assay quantification from supernatant obtained from infected podocytes at 24, 48 and 72 h.p.i. (E) qPCR analysis of podocyte-specific genes revealed that both synaptopodin (SYNPO) and podocalyxin (PODXL) are significantly upregulated after infection with SARS-CoV-2 at MOI of 0.01, whereas SYNPO is significantly upregulated at multiple MOIs, and PODXL shows no significant changes with viral infection. (F) The expression of spike-associated genes (ACE2, BSG/CD147) and spike processing genes (TMPRSS2, CTSL) are significantly impacted by infection at MOIs of 0.01 and 0.1, respectively. (G) Human iPS cell-derived podocytes treated with SARS-CoV-2 (at MOI of 0.01) immunostain positive for Nucleocapsid protein (magenta), indicating successful infection with the virus. The cells were immunostained also for the podocyte marker Nephrin (green) and counterstained with DAPI (blue). Scale bar: 100 µm (H) Spike positive cells Nephrin and DAPI as nuclear counterstain in the infected podocytes. Scale bar: 100 µm. One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p

    Techniques Used: Derivative Assay, Infection, Real-time Polymerase Chain Reaction, Plaque Assay, Expressing, Marker

    Transcriptomic and protein level analyses of spike-interacting factors in mature podocytes differentiated from human iPS cells. (A) Heatmap showing expression levels of spike associated genes from three biological human iPS cell-derived podocyte samples. SIGLEC9, Sialic acid-binding Ig-like lectin 9; CAPZA1, F-actin-capping protein subunit alpha-1; CLEC10A, C-type lectin domain family 10 member A; CD33, Myeloid cell surface antigen CD33; TMOD3, Tropomodulin-3; ACE2, Angiotensin Converting Enzyme 2; BSG/CD147, Basigin/CD147 molecule; CD209, CD209 Antigen; MYO6, Unconventional myosin-VI; PLS3, Plastin-3; LDHB, L-lactate dehydrogenase B chain; GNB2L1/RACK, Receptor of activated protein C kinase 1; SNRNP70, U1 small nuclear ribonucleoprotein 70 kDa; DOCK7, Dedicator of cytokinesis protein 7; RPS18, 40S ribosomal protein S18; CAPZB, F-actin-capping protein subunit beta; GOLGA7, Golgin subfamily A member 7; ZDHHC5, Palmitoyltransferase ZDHHC5; SIGLEC10, Sialic acid-binding Ig-like lectin 10; ACTR3, Actin-related protein 3; MYL6, Myosin light polypeptide 6; CORO1C, Coronin-1C; ARPC4, Actin-related protein 2/3 complex subunit 4; CCT6A, T-complex protein 1 subunit zeta; (B) qPCR quantification of nine of the human spike-associated gene from (A) including Transmembrane Serine Protease 2 (TMPRSS2) or cathepsin L (CTSL) in human iPS cell-derived podocytes, Calu-3, Caco-2, glomerular endothelial cells, and HEK 293T cells (normalized to Calu-3 groups). (C) Western blot analysis to evaluate protein expression of ACE2, BSG/CD147, CD209, TMPRSS2 and CTSL in the different cell type used; Calu-3, human iPS cell-derived podocytes, Caco-2, glomerular endothelial (gEndos) cells, and HEK 293T cells. (D) Immunocytochemistry analysis of ACE2, BSG/CD147, CD209, TMPRSS2 and CTSL expression in iPS cell-derived podocytes. Scale bar: 100 µm. One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p
    Figure Legend Snippet: Transcriptomic and protein level analyses of spike-interacting factors in mature podocytes differentiated from human iPS cells. (A) Heatmap showing expression levels of spike associated genes from three biological human iPS cell-derived podocyte samples. SIGLEC9, Sialic acid-binding Ig-like lectin 9; CAPZA1, F-actin-capping protein subunit alpha-1; CLEC10A, C-type lectin domain family 10 member A; CD33, Myeloid cell surface antigen CD33; TMOD3, Tropomodulin-3; ACE2, Angiotensin Converting Enzyme 2; BSG/CD147, Basigin/CD147 molecule; CD209, CD209 Antigen; MYO6, Unconventional myosin-VI; PLS3, Plastin-3; LDHB, L-lactate dehydrogenase B chain; GNB2L1/RACK, Receptor of activated protein C kinase 1; SNRNP70, U1 small nuclear ribonucleoprotein 70 kDa; DOCK7, Dedicator of cytokinesis protein 7; RPS18, 40S ribosomal protein S18; CAPZB, F-actin-capping protein subunit beta; GOLGA7, Golgin subfamily A member 7; ZDHHC5, Palmitoyltransferase ZDHHC5; SIGLEC10, Sialic acid-binding Ig-like lectin 10; ACTR3, Actin-related protein 3; MYL6, Myosin light polypeptide 6; CORO1C, Coronin-1C; ARPC4, Actin-related protein 2/3 complex subunit 4; CCT6A, T-complex protein 1 subunit zeta; (B) qPCR quantification of nine of the human spike-associated gene from (A) including Transmembrane Serine Protease 2 (TMPRSS2) or cathepsin L (CTSL) in human iPS cell-derived podocytes, Calu-3, Caco-2, glomerular endothelial cells, and HEK 293T cells (normalized to Calu-3 groups). (C) Western blot analysis to evaluate protein expression of ACE2, BSG/CD147, CD209, TMPRSS2 and CTSL in the different cell type used; Calu-3, human iPS cell-derived podocytes, Caco-2, glomerular endothelial (gEndos) cells, and HEK 293T cells. (D) Immunocytochemistry analysis of ACE2, BSG/CD147, CD209, TMPRSS2 and CTSL expression in iPS cell-derived podocytes. Scale bar: 100 µm. One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p

    Techniques Used: Expressing, Derivative Assay, Binding Assay, Real-time Polymerase Chain Reaction, Western Blot, Immunocytochemistry

    Antibody blocking reveal roles of ACE2 and BSG/CD147 receptors in viral uptake. (A) qPCR quantification of S-pseudotyped entry relative to antibody concentration (normalized to unblocked samples). Human iPS cell-derived podocytes were incubated with different dilutions of anti-ACE2, anti-BSG or both for an hour followed by infection with S-pseudotyped virus at MOI 0.02 for 60 h (B) qPCR quantification of Spike binding receptor genes (ACE2, BSG/CD147) and Spike processing factor genes (TMPRSS2, CTSL) at 5 µg/ml for both anti-ACE2 antibody blockage and anti-BSG antibody blockage showing significant changes in gene expression with optimal receptor blockage when compared to unblocked samples (normalized to mock groups). One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p
    Figure Legend Snippet: Antibody blocking reveal roles of ACE2 and BSG/CD147 receptors in viral uptake. (A) qPCR quantification of S-pseudotyped entry relative to antibody concentration (normalized to unblocked samples). Human iPS cell-derived podocytes were incubated with different dilutions of anti-ACE2, anti-BSG or both for an hour followed by infection with S-pseudotyped virus at MOI 0.02 for 60 h (B) qPCR quantification of Spike binding receptor genes (ACE2, BSG/CD147) and Spike processing factor genes (TMPRSS2, CTSL) at 5 µg/ml for both anti-ACE2 antibody blockage and anti-BSG antibody blockage showing significant changes in gene expression with optimal receptor blockage when compared to unblocked samples (normalized to mock groups). One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p

    Techniques Used: Blocking Assay, Real-time Polymerase Chain Reaction, Concentration Assay, Derivative Assay, Incubation, Infection, Binding Assay, Expressing

    32) Product Images from "Bromodomain factor 5 is an essential regulator of transcription in Leishmania"

    Article Title: Bromodomain factor 5 is an essential regulator of transcription in Leishmania

    Journal: Nature Communications

    doi: 10.1038/s41467-022-31742-1

    Effect of BDF5 depletion on RNA levels and gene expression. a Flow cytometry of cells stained with SYTO RNASelect Stain to measure total RNA levels in Lmx::DiCre strains or the BDF5 −/+flx strain treated with rapamycin or DMSO over a 72 h time course. 20,000 events measured per condition. b Dot plot of total RNA-seq reads per protein-coding gene scaled to ERCC spike-in controls, then as a percentage of the DMSO control sample, separated per chromosome, conducted at a 96 h timepoint. Black lines denote the median of the scaled response for each chromosome, individual data points are means of 2 separate RNA seq experiments, the number of CDS features quantified on each chromosome is indicated above the dot plots. c Metaplot of divergent SSR ( n = 60) for DMSO treated or rapamycin-treated BDF5 −/+flx showing combined reads from the positive and negative strands. d . Metaplot of reads mapping to the + strand, normalised to ERCC control at divergent SSRs ( n = 60) of DMSO treated or rapamycin-treated BDF5 −/+flx cultures. e Metaplot of + stranded RNA-seq reads normalised to ERCC spike-in controls for PTUs ( n = 120), on a scale of 0–100%. f . Metaplot of reads mapping to the + and − strands, normalised to ERCC control at convergent SSRs ( n = 40) of DMSO treated or rapamycin-treated BDF5 −/+flx cultures. Metaplot data is from 1 representative of the three replicate RNA-seq datasets. g Spike-in controlled SYBR RT-qPCR of reporter genes for Pol I, II, III. BDF5 deletion was induced for 96 h and total RNA was extracted with lysis buffer spiked with yeast total RNA to provide a normalisation channel using a primer set against yeast actin, allowing comparison of the relative 18s rRNA, Cyclophilin A, and tRNA Lys RNA levels compared to DMSO treated cells. Bars denote mean, error bars denote standard deviation. Comparisons by multiple two-sided t test, corrected with Benjamini and Hochberg method, p-values indicate above, * denotes a discovery, n = 5 replicate PCR reactions. ACT1 values were not compared as this was the normalisation target.
    Figure Legend Snippet: Effect of BDF5 depletion on RNA levels and gene expression. a Flow cytometry of cells stained with SYTO RNASelect Stain to measure total RNA levels in Lmx::DiCre strains or the BDF5 −/+flx strain treated with rapamycin or DMSO over a 72 h time course. 20,000 events measured per condition. b Dot plot of total RNA-seq reads per protein-coding gene scaled to ERCC spike-in controls, then as a percentage of the DMSO control sample, separated per chromosome, conducted at a 96 h timepoint. Black lines denote the median of the scaled response for each chromosome, individual data points are means of 2 separate RNA seq experiments, the number of CDS features quantified on each chromosome is indicated above the dot plots. c Metaplot of divergent SSR ( n = 60) for DMSO treated or rapamycin-treated BDF5 −/+flx showing combined reads from the positive and negative strands. d . Metaplot of reads mapping to the + strand, normalised to ERCC control at divergent SSRs ( n = 60) of DMSO treated or rapamycin-treated BDF5 −/+flx cultures. e Metaplot of + stranded RNA-seq reads normalised to ERCC spike-in controls for PTUs ( n = 120), on a scale of 0–100%. f . Metaplot of reads mapping to the + and − strands, normalised to ERCC control at convergent SSRs ( n = 40) of DMSO treated or rapamycin-treated BDF5 −/+flx cultures. Metaplot data is from 1 representative of the three replicate RNA-seq datasets. g Spike-in controlled SYBR RT-qPCR of reporter genes for Pol I, II, III. BDF5 deletion was induced for 96 h and total RNA was extracted with lysis buffer spiked with yeast total RNA to provide a normalisation channel using a primer set against yeast actin, allowing comparison of the relative 18s rRNA, Cyclophilin A, and tRNA Lys RNA levels compared to DMSO treated cells. Bars denote mean, error bars denote standard deviation. Comparisons by multiple two-sided t test, corrected with Benjamini and Hochberg method, p-values indicate above, * denotes a discovery, n = 5 replicate PCR reactions. ACT1 values were not compared as this was the normalisation target.

    Techniques Used: Expressing, Flow Cytometry, Staining, RNA Sequencing Assay, Quantitative RT-PCR, Lysis, Standard Deviation, Polymerase Chain Reaction

    33) Product Images from "Genetic Factors and Genotype-Environment Interactions Contribute to Variation in Melanin Production in the Fungal Pathogen Cryptococcus neoformans"

    Article Title: Genetic Factors and Genotype-Environment Interactions Contribute to Variation in Melanin Production in the Fungal Pathogen Cryptococcus neoformans

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-27813-3

    Normalized LAC1 expression in low, intermediate and high nitrosative stress conditions. The abundance of LAC1 mRNA transcripts was quantified in two representative strains from the study population using RT-qPCR. In both strains, the normalized LAC1 expression was correlated with the corresponding level of melanin production. LAC1 expression was induced in intermediate nitrosative stress in comparison to low and high nitrosative stress.
    Figure Legend Snippet: Normalized LAC1 expression in low, intermediate and high nitrosative stress conditions. The abundance of LAC1 mRNA transcripts was quantified in two representative strains from the study population using RT-qPCR. In both strains, the normalized LAC1 expression was correlated with the corresponding level of melanin production. LAC1 expression was induced in intermediate nitrosative stress in comparison to low and high nitrosative stress.

    Techniques Used: Expressing, Quantitative RT-PCR

    34) Product Images from "A Biomimetic Electrospun Membrane Supports the Differentiation and Maturation of Kidney Epithelium from Human Stem Cells"

    Article Title: A Biomimetic Electrospun Membrane Supports the Differentiation and Maturation of Kidney Epithelium from Human Stem Cells

    Journal: Bioengineering

    doi: 10.3390/bioengineering9050188

    Quantification of podocyte-specific genes and proteins expressed in cells differentiated on electrospun silk fibroin. ( a ) RT-qPCR quantification of podocyte-specific genes. Yellow, podocytes differentiated on plastic culture plates; Red, podocytes differentiated on electrospun silk fibroin membrane. N = 3 independent experiments. ns, non-significant. * p
    Figure Legend Snippet: Quantification of podocyte-specific genes and proteins expressed in cells differentiated on electrospun silk fibroin. ( a ) RT-qPCR quantification of podocyte-specific genes. Yellow, podocytes differentiated on plastic culture plates; Red, podocytes differentiated on electrospun silk fibroin membrane. N = 3 independent experiments. ns, non-significant. * p

    Techniques Used: Quantitative RT-PCR

    35) Product Images from "Hfq CLASH uncovers sRNA-target interaction networks involved in adaptation to nutrient availability"

    Article Title: Hfq CLASH uncovers sRNA-target interaction networks involved in adaptation to nutrient availability

    Journal: bioRxiv

    doi: 10.1101/481986

    sRNA-RNA interactions identified by CLASH are growth-stage specific. ( A ) Hfq CLASH uncovers sRNA-sRNA networks: comparison between statistically filtered sRNA-sRNA interactions in the Hfq CLASH data, RIL-seq S-chimeras ( Melamed et al., 2016 ) (log and stationary) and RNase E CLASH ( Waters et al., 2016 ). Only core genome sRNAs were considered. Red coloured sRNA-sRNA interactions have been characterized in more detail. ( B ) Heatmaps showing the read density (log2(chimera count)) of chimeric fragments mapping to ArcZ and CyaR. (Top) ArcZ regions involved in sRNA-sRNA interactions. The location of the known ArcZ seed sequence is indicated above. (Bottom) CyaR heatmaps that show all CyaR chimeras and CyaR-sRNA chimeras, respectively. The location of the known CyaR seed sequence, as well as a new seed, is indicated above. ( C ) Base-pairing interactions predicted from the ArcZ-CyaR chimeras using RNAcofold. The nucleotide substitutions for experimental validation of direct base-pairing are shown as red or green residues. ( D ) SRNA-sRNA interactions coordinate nutritional stress responses. ArcZ, CyaR were overexpressed and the levels of their targets were monitored by RT-qPCR. The tpx and sdaC mRNAs are ArcZ mRNA targets. The nadE and yqaE mRNAs are CyaR targets. The dppA mRNA is a GcvB target. Experiments were performed in biological and technical triplicates; Error bars indicate the standard error of the mean (SEM) of the three biological replicates. The dashed horizontal line indicates the level of the overexpressed scrambled RNA. ( E ) ArcZ and CyaR directly interact. The sRNAs and mutants as in ( C ) were ectopically co-expressed in E. coli and CyaR and CyaR 38-39 levels were quantified by RT-qPCR.
    Figure Legend Snippet: sRNA-RNA interactions identified by CLASH are growth-stage specific. ( A ) Hfq CLASH uncovers sRNA-sRNA networks: comparison between statistically filtered sRNA-sRNA interactions in the Hfq CLASH data, RIL-seq S-chimeras ( Melamed et al., 2016 ) (log and stationary) and RNase E CLASH ( Waters et al., 2016 ). Only core genome sRNAs were considered. Red coloured sRNA-sRNA interactions have been characterized in more detail. ( B ) Heatmaps showing the read density (log2(chimera count)) of chimeric fragments mapping to ArcZ and CyaR. (Top) ArcZ regions involved in sRNA-sRNA interactions. The location of the known ArcZ seed sequence is indicated above. (Bottom) CyaR heatmaps that show all CyaR chimeras and CyaR-sRNA chimeras, respectively. The location of the known CyaR seed sequence, as well as a new seed, is indicated above. ( C ) Base-pairing interactions predicted from the ArcZ-CyaR chimeras using RNAcofold. The nucleotide substitutions for experimental validation of direct base-pairing are shown as red or green residues. ( D ) SRNA-sRNA interactions coordinate nutritional stress responses. ArcZ, CyaR were overexpressed and the levels of their targets were monitored by RT-qPCR. The tpx and sdaC mRNAs are ArcZ mRNA targets. The nadE and yqaE mRNAs are CyaR targets. The dppA mRNA is a GcvB target. Experiments were performed in biological and technical triplicates; Error bars indicate the standard error of the mean (SEM) of the three biological replicates. The dashed horizontal line indicates the level of the overexpressed scrambled RNA. ( E ) ArcZ and CyaR directly interact. The sRNAs and mutants as in ( C ) were ectopically co-expressed in E. coli and CyaR and CyaR 38-39 levels were quantified by RT-qPCR.

    Techniques Used: Sequencing, Quantitative RT-PCR

    MdoR regulates ompC mRNA translation in E. coli . (A) Cultures at OD 600 0.4 overexpressing MdoR or no sRNA for 15 minutes were subjected to polysome profiling. Profiles of the polysomal (2×70S) and subpolysomal fractions obtained for the empty plasmid control and MdoR overexpression samples. (B) RT-qPCR analysis of the polysomal fractions: A ‘total’ fraction was obtained by mixing equal amounts/volumes of the polysomal and subpolysomal fractions and is representative of the cytosol/cell lysate content. Total RNA was extracted from all fractions (polysomal, subpolysomal and total). Expression of ompC in the polysomal fractions was quantified relative to the amount in ‘total’ fraction, normalized to recA , and calculated as fold-change relative to the control sample (y-axis). The experiments were performed in technical triplicates; the standard error of the mean (SEM) of three biological replicates fold changes are reported as error bars. Significance of the difference in ompC mRNA level in polysomes was assessed with a two-tailed Student’s t-test. Source data for are provided as a Source Data file.
    Figure Legend Snippet: MdoR regulates ompC mRNA translation in E. coli . (A) Cultures at OD 600 0.4 overexpressing MdoR or no sRNA for 15 minutes were subjected to polysome profiling. Profiles of the polysomal (2×70S) and subpolysomal fractions obtained for the empty plasmid control and MdoR overexpression samples. (B) RT-qPCR analysis of the polysomal fractions: A ‘total’ fraction was obtained by mixing equal amounts/volumes of the polysomal and subpolysomal fractions and is representative of the cytosol/cell lysate content. Total RNA was extracted from all fractions (polysomal, subpolysomal and total). Expression of ompC in the polysomal fractions was quantified relative to the amount in ‘total’ fraction, normalized to recA , and calculated as fold-change relative to the control sample (y-axis). The experiments were performed in technical triplicates; the standard error of the mean (SEM) of three biological replicates fold changes are reported as error bars. Significance of the difference in ompC mRNA level in polysomes was assessed with a two-tailed Student’s t-test. Source data for are provided as a Source Data file.

    Techniques Used: Plasmid Preparation, Over Expression, Quantitative RT-PCR, Expressing, Two Tailed Test

    36) Product Images from "Identification of a GrgA-Euo-HrcA Transcriptional Regulatory Network in Chlamydia"

    Article Title: Identification of a GrgA-Euo-HrcA Transcriptional Regulatory Network in Chlamydia

    Journal: mSystems

    doi: 10.1128/mSystems.00738-21

    Genome copy numbers and temporary expression patterns of endogenous GrgA, Euo, and HrcA during the CtL2 developmental cycle. L929 cells infected with wild-type CtL2 at the multiplicity of infection of 0.5 inclusion-forming unit per cell were harvested at 0, 1, 3, 5, 8, 12, 18, 24, 30, and 36 hpi. Genomic DNA (gDNA) was quantified using qPCR (biological triplicates). RNAs of grgA , euo , and hrcA were quantified using qRT-PCR (biological triplicates). GrgA protein was quantified using ELISA (biological duplicates). All expression data were normalized with the amount of gDNA. See Fig. S5 for genome copy number data and expression results of individual genes with error bars.
    Figure Legend Snippet: Genome copy numbers and temporary expression patterns of endogenous GrgA, Euo, and HrcA during the CtL2 developmental cycle. L929 cells infected with wild-type CtL2 at the multiplicity of infection of 0.5 inclusion-forming unit per cell were harvested at 0, 1, 3, 5, 8, 12, 18, 24, 30, and 36 hpi. Genomic DNA (gDNA) was quantified using qPCR (biological triplicates). RNAs of grgA , euo , and hrcA were quantified using qRT-PCR (biological triplicates). GrgA protein was quantified using ELISA (biological duplicates). All expression data were normalized with the amount of gDNA. See Fig. S5 for genome copy number data and expression results of individual genes with error bars.

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Effects of overexpression of GrgA forms, Euo and HrcA on CtL2 growth in L929 cells. (A) Differential effects of 10 nM ATC treatment on inclusion morphology and RFP intensity in cultures of CtL2/GrgA, CtL2/GrgAΔσ 66 BD, and CtL2/GrgAΔσ 28 BD. ATC was added at 0 hpi. Images were acquired at 35 hpi. (B) Genome copy reduction in CtL2/GrgA after treatment with 10 nM ATC for 4 h (from 12 to 16 hpi). Genome copy was determined using qPCR. (C) Moderate increases in euo and hrcA expression in CtL2/Euo and CtL2/HrcA, respectively, after treatment with a low concentration of ATC for 4 h (from 12 to 16 hpi). RNAs of euo and hrcA were quantified using qRT-PCR. Note that mRNA expression values have been normalized with gDNA due to (possible) overexpression-induced growth inhibition (D) Genome copy reduction in CtL2/Euo but not CtL2/HrcA after treatment with a low concentration of ATC for 4 h. Genome copy was determined using qPCR. In panels B to D, all quantitative data were obtained with biological triplicates.
    Figure Legend Snippet: Effects of overexpression of GrgA forms, Euo and HrcA on CtL2 growth in L929 cells. (A) Differential effects of 10 nM ATC treatment on inclusion morphology and RFP intensity in cultures of CtL2/GrgA, CtL2/GrgAΔσ 66 BD, and CtL2/GrgAΔσ 28 BD. ATC was added at 0 hpi. Images were acquired at 35 hpi. (B) Genome copy reduction in CtL2/GrgA after treatment with 10 nM ATC for 4 h (from 12 to 16 hpi). Genome copy was determined using qPCR. (C) Moderate increases in euo and hrcA expression in CtL2/Euo and CtL2/HrcA, respectively, after treatment with a low concentration of ATC for 4 h (from 12 to 16 hpi). RNAs of euo and hrcA were quantified using qRT-PCR. Note that mRNA expression values have been normalized with gDNA due to (possible) overexpression-induced growth inhibition (D) Genome copy reduction in CtL2/Euo but not CtL2/HrcA after treatment with a low concentration of ATC for 4 h. Genome copy was determined using qPCR. In panels B to D, all quantitative data were obtained with biological triplicates.

    Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Expressing, Concentration Assay, Quantitative RT-PCR, Inhibition

    37) Product Images from "Roles of Argonautes and Dicers on Sclerotinia sclerotiorum Antiviral RNA Silencing"

    Article Title: Roles of Argonautes and Dicers on Sclerotinia sclerotiorum Antiviral RNA Silencing

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2019.00976

    Effects of external RNA pesticide on inhibiting S. sclerotiorum from causing lesions on canola leaves comparing dsRNA targeting agl -2 at 800 ng/20 μl, 400 ng/20 μl, 200 ng/20 μl, dsRNA targeting agl -4 at 800 ng/20 μl as control (from left to right), confirmed by (A) RT-qPCR with reduced expression levels of agl -2 at 200 and 400 ng, and lesion comparison at (B) 36, (C) 48, and (D) 72 h post-inoculation.
    Figure Legend Snippet: Effects of external RNA pesticide on inhibiting S. sclerotiorum from causing lesions on canola leaves comparing dsRNA targeting agl -2 at 800 ng/20 μl, 400 ng/20 μl, 200 ng/20 μl, dsRNA targeting agl -4 at 800 ng/20 μl as control (from left to right), confirmed by (A) RT-qPCR with reduced expression levels of agl -2 at 200 and 400 ng, and lesion comparison at (B) 36, (C) 48, and (D) 72 h post-inoculation.

    Techniques Used: Quantitative RT-PCR, Expressing

    38) Product Images from "Modulating the evolutionary trajectory of tolerance using antibiotics with different metabolic dependencies"

    Article Title: Modulating the evolutionary trajectory of tolerance using antibiotics with different metabolic dependencies

    Journal: Nature Communications

    doi: 10.1038/s41467-022-30272-0

    A novel mechanism of tolerance through nhaA loss. a Deletion of an ~2.6–2.7 kb region from the E. coli BW25113 genome, including the sodium-proton antiporter nhaA and its regulator nhaR , in all replicates of ampicillin-evolved cells was identified through whole-genome sequencing. One replicate also had a 1 bp insertion near the IS1A insertion element. b Percent survival of ancestral, amp D8, ΔnhaA , and Δ nhaR under ampicillin treatment. Overnight cultures were diluted 1:100 and treated with ampicillin for 6 hours. Shown is the mean of three biological replicates; error bars represent SEM. c Relative Δ nhaA fitness under ampicillin treatment and an untreated control. Overnight cultures of ancestral and Δ nhaA were combined and diluted 1:100 into fresh LB, then treated with ampicillin or a vehicle control (water) for 6 hours. The mean of three biological replicates is shown; error bars indicate SEM. d Hierarchical clustering of differentially expressed genes (DEGs) for three biological replicates of ancestral and amp D8. Heat map color shows z-score normalized counts per million (CPM). DEGs belonging to each GO term category are denoted on the left and in the bottom legend. GO term categories included are cell adhesion involved in biofilm formation (GO:0043708), SOS response (GO:0009432), glycolytic process (GO:0006096), ATP metabolic process (GO:0046034), tricarboxylic acid cycle (GO:0006099), and aerobic respiration (GO:0009060). e Intracellular ATP concentration in ancestral, amp D8, and ΔnhaA . Data are representative of three biological replicates; error bars denote SEM. Source data are provided as a Source Data file.
    Figure Legend Snippet: A novel mechanism of tolerance through nhaA loss. a Deletion of an ~2.6–2.7 kb region from the E. coli BW25113 genome, including the sodium-proton antiporter nhaA and its regulator nhaR , in all replicates of ampicillin-evolved cells was identified through whole-genome sequencing. One replicate also had a 1 bp insertion near the IS1A insertion element. b Percent survival of ancestral, amp D8, ΔnhaA , and Δ nhaR under ampicillin treatment. Overnight cultures were diluted 1:100 and treated with ampicillin for 6 hours. Shown is the mean of three biological replicates; error bars represent SEM. c Relative Δ nhaA fitness under ampicillin treatment and an untreated control. Overnight cultures of ancestral and Δ nhaA were combined and diluted 1:100 into fresh LB, then treated with ampicillin or a vehicle control (water) for 6 hours. The mean of three biological replicates is shown; error bars indicate SEM. d Hierarchical clustering of differentially expressed genes (DEGs) for three biological replicates of ancestral and amp D8. Heat map color shows z-score normalized counts per million (CPM). DEGs belonging to each GO term category are denoted on the left and in the bottom legend. GO term categories included are cell adhesion involved in biofilm formation (GO:0043708), SOS response (GO:0009432), glycolytic process (GO:0006096), ATP metabolic process (GO:0046034), tricarboxylic acid cycle (GO:0006099), and aerobic respiration (GO:0009060). e Intracellular ATP concentration in ancestral, amp D8, and ΔnhaA . Data are representative of three biological replicates; error bars denote SEM. Source data are provided as a Source Data file.

    Techniques Used: Sequencing, Concentration Assay

    SDM/WDM cycling delays evolution of SDM tolerance. a Schematic of antibiotic cycling evolutions. Evolutions were conducted identically to Fig. 1 , except the applied antibiotic was cycled as indicated. b Percent survival from evolutions where two antibiotics were alternated every day. Horizontal axis labels indicate the antibiotic(s) on which the culture was evolved and the evolution day. Survival assays were conducted with ampicillin and ciprofloxacin; survival data are grouped by the antibiotic used for phenotyping. Data are representative of three biological replicates; error bars indicate SEM. c MICs of evolutions where two antibiotics were alternated every day. Three biological replicates were assayed; error bars indicate SEM. d Predicted number of tolerant mutant cells after the growth phase of each evolution day for various ampicillin/mitomycin C cycling regimens. One thousand simulations of the model were run and the output from a simulation with the median takeover day is shown. The mutation rate parameter used for these simulations was 50 × 10 −10 mutations per division 3 . e Violin plot representation of the mutant takeover day over 1000 model simulations. Mutant takeover day is defined as when the number of tolerant mutant cells exceeds the number of wild-type cells. Simulation values of 120 and greater are binned together. Solid white lines indicate the median simulation value and dotted lines represent the 25 th and 75 th percentiles. The width of each shaded area is indicative of the frequency of that value amongst the 1000 simulations, and the length of the shaded area extends from the minimum to maximum simulation value. The mutation rate parameter used for these simulations was 50 × 10 −10 mutations per division 3 . f Experimental percent survival for evolutions conducted with all ampicillin, all mitomycin C, or cycling of 1–4 days of ampicillin treatment with 1 day of mitomycin C treatment. Survival assays with 50 µg/mL ampicillin were done on days 10, 20, and 30. Data are representative of three biological replicates; error bars denote SEM. g Ampicillin MIC for cycling regimens conducted with all ampicillin, all mitomycin C, or cycling of 1–4 days of ampicillin treatment with 1 day of mitomycin C treatment. MICs were taken from days 10, 20, and 30 of each evolution. Data are representative of three biological replicates; error bars represent SEM. Source data are provided as a Source Data file.
    Figure Legend Snippet: SDM/WDM cycling delays evolution of SDM tolerance. a Schematic of antibiotic cycling evolutions. Evolutions were conducted identically to Fig. 1 , except the applied antibiotic was cycled as indicated. b Percent survival from evolutions where two antibiotics were alternated every day. Horizontal axis labels indicate the antibiotic(s) on which the culture was evolved and the evolution day. Survival assays were conducted with ampicillin and ciprofloxacin; survival data are grouped by the antibiotic used for phenotyping. Data are representative of three biological replicates; error bars indicate SEM. c MICs of evolutions where two antibiotics were alternated every day. Three biological replicates were assayed; error bars indicate SEM. d Predicted number of tolerant mutant cells after the growth phase of each evolution day for various ampicillin/mitomycin C cycling regimens. One thousand simulations of the model were run and the output from a simulation with the median takeover day is shown. The mutation rate parameter used for these simulations was 50 × 10 −10 mutations per division 3 . e Violin plot representation of the mutant takeover day over 1000 model simulations. Mutant takeover day is defined as when the number of tolerant mutant cells exceeds the number of wild-type cells. Simulation values of 120 and greater are binned together. Solid white lines indicate the median simulation value and dotted lines represent the 25 th and 75 th percentiles. The width of each shaded area is indicative of the frequency of that value amongst the 1000 simulations, and the length of the shaded area extends from the minimum to maximum simulation value. The mutation rate parameter used for these simulations was 50 × 10 −10 mutations per division 3 . f Experimental percent survival for evolutions conducted with all ampicillin, all mitomycin C, or cycling of 1–4 days of ampicillin treatment with 1 day of mitomycin C treatment. Survival assays with 50 µg/mL ampicillin were done on days 10, 20, and 30. Data are representative of three biological replicates; error bars denote SEM. g Ampicillin MIC for cycling regimens conducted with all ampicillin, all mitomycin C, or cycling of 1–4 days of ampicillin treatment with 1 day of mitomycin C treatment. MICs were taken from days 10, 20, and 30 of each evolution. Data are representative of three biological replicates; error bars represent SEM. Source data are provided as a Source Data file.

    Techniques Used: Mutagenesis

    39) Product Images from "Mouse B2 SINE elements function as IFN-inducible enhancers"

    Article Title: Mouse B2 SINE elements function as IFN-inducible enhancers

    Journal: bioRxiv

    doi: 10.1101/2022.08.02.502523

    B2_Mm2 in the genomic landscape. (A) RT-qPCR of clonal replicates for WT (n=3) J774A.1 cells and J774A.1 cells harboring the B2_Mm2.Dicer1 deletion (n=2) measuring Dicer1 expression relative to CTCF . Treatments are indicated by color. * p -value
    Figure Legend Snippet: B2_Mm2 in the genomic landscape. (A) RT-qPCR of clonal replicates for WT (n=3) J774A.1 cells and J774A.1 cells harboring the B2_Mm2.Dicer1 deletion (n=2) measuring Dicer1 expression relative to CTCF . Treatments are indicated by color. * p -value

    Techniques Used: Quantitative RT-PCR, Expressing

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    New England Biolabs luna universal one step rt qpcr kit
    Screening of compounds with antiviral activity targeting <t>SARS-CoV-2</t> host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by <t>RT-qPCR</t> (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p
    Luna Universal One Step Rt Qpcr Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs luna universal probe one step rt qpcr kit
    Mitoprotein-induced stress triggers the UPR ER . A , Fusion of DHFR to the N-terminus of Cytochrome b 2 generates a mitochondrial ‘clogger’ that jams the protein import machinery. B , The mitochondrial clogger b 2 -DHFR or cytosolic DHFR were expressed for 4.5 h. The precursor form of the mitochondrial proteins Mdj1 and Rip1 were detected by Western Blotting. C , Expression of b 2 -DHFR leads to attenuated growth. D , The mitoprotein-induced stress response encompasses an early transcriptional induction of chaperones and the proteasome and a downregulation of cytosolic ribosomes and OXPHOS components. E , Protein levels in clogger-expressing versus control cells after 18 h of induction were measured by quantitative mass spectrometry 28 . Highlighted are proteins which are reported targets of the UPR ER 21 . Data from n =3 independent biological replicates are shown. F, G , The cellular transcriptome and translatome after 4.5 h of clogger induction were measured by <t>RNA-Seq</t> ( n =4) 28 and ribosome profiling ( n =3), respectively. Shown are log 2 fold changes of b 2 -DHFR versus cytosolic DHFR. HAC1 transcripts are slightly reduced, but its translation is upregulated. H , Ribosome footprints along the HAC1 gene from cells expressing b 2 -DHFR or cytosolic DHFR for 4.5 h are shown. I , Levels of spliced HAC1 mRNA in cells expressing b 2 -DHFR or cytosolic DHFR were measured by <t>RT-qPCR</t> over time ( n =3).
    Luna Universal Probe One Step Rt Qpcr Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Screening of compounds with antiviral activity targeting SARS-CoV-2 host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p

    Journal: bioRxiv

    Article Title: Characterization and functional interrogation of SARS-CoV-2 RNA interactome

    doi: 10.1101/2021.03.23.436611

    Figure Lengend Snippet: Screening of compounds with antiviral activity targeting SARS-CoV-2 host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p

    Article Snippet: Yields of viral RNA were quantified by real-time qPCR by using SARS-CoV-2 specific primers targeting the E gene with the Luna®Universal One-Step RT-qPCR Kit (New England Biolabs) in a LightCycler 480 thermocycler (Roche) according to the manufacturer’s protocol.

    Techniques: Activity Assay, Infection, Quantitative RT-PCR

    Functional interrogation of the SARS-CoV-2 RNA interactome and compounds screening. (A) Schematic illustrating the loss-of-function screen procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles released in the supernatant of infected cells was quantified by RT-qPCR and normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data shown are means of two independent experiments. Adjusted p-values were calculated by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors are marked in blue and host restriction factors are marked in red. Positive controls (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained from N protein quantification by flow cytometry and virus release in supernatant of infected cells by RT-qPCR. Data shown are means of two independent experiments. Host dependency factors are marked in blue and host restriction factors are marked in red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown are mean +/- SD of three independent experiments in duplicate.

    Journal: bioRxiv

    Article Title: Characterization and functional interrogation of SARS-CoV-2 RNA interactome

    doi: 10.1101/2021.03.23.436611

    Figure Lengend Snippet: Functional interrogation of the SARS-CoV-2 RNA interactome and compounds screening. (A) Schematic illustrating the loss-of-function screen procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles released in the supernatant of infected cells was quantified by RT-qPCR and normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data shown are means of two independent experiments. Adjusted p-values were calculated by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors are marked in blue and host restriction factors are marked in red. Positive controls (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained from N protein quantification by flow cytometry and virus release in supernatant of infected cells by RT-qPCR. Data shown are means of two independent experiments. Host dependency factors are marked in blue and host restriction factors are marked in red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown are mean +/- SD of three independent experiments in duplicate.

    Article Snippet: Yields of viral RNA were quantified by real-time qPCR by using SARS-CoV-2 specific primers targeting the E gene with the Luna®Universal One-Step RT-qPCR Kit (New England Biolabs) in a LightCycler 480 thermocycler (Roche) according to the manufacturer’s protocol.

    Techniques: Functional Assay, Transfection, Infection, Quantitative RT-PCR, Flow Cytometry

    Susceptibility of human iPS cell-derived podocytes to infection by live SARS-CoV-2. qPCR analysis of human iPS cell-derived podocytes infected with SARS-CoV-2 revealed intracellular uptake of the virus for 24 h.p.i (A) , 48 h.p.i (B) and 72 h.p.i (C) . (D) plaque assay quantification from supernatant obtained from infected podocytes at 24, 48 and 72 h.p.i. (E) qPCR analysis of podocyte-specific genes revealed that both synaptopodin (SYNPO) and podocalyxin (PODXL) are significantly upregulated after infection with SARS-CoV-2 at MOI of 0.01, whereas SYNPO is significantly upregulated at multiple MOIs, and PODXL shows no significant changes with viral infection. (F) The expression of spike-associated genes (ACE2, BSG/CD147) and spike processing genes (TMPRSS2, CTSL) are significantly impacted by infection at MOIs of 0.01 and 0.1, respectively. (G) Human iPS cell-derived podocytes treated with SARS-CoV-2 (at MOI of 0.01) immunostain positive for Nucleocapsid protein (magenta), indicating successful infection with the virus. The cells were immunostained also for the podocyte marker Nephrin (green) and counterstained with DAPI (blue). Scale bar: 100 µm (H) Spike positive cells Nephrin and DAPI as nuclear counterstain in the infected podocytes. Scale bar: 100 µm. One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: SARS-CoV-2 Employ BSG/CD147 and ACE2 Receptors to Directly Infect Human Induced Pluripotent Stem Cell-Derived Kidney Podocytes

    doi: 10.3389/fcell.2022.855340

    Figure Lengend Snippet: Susceptibility of human iPS cell-derived podocytes to infection by live SARS-CoV-2. qPCR analysis of human iPS cell-derived podocytes infected with SARS-CoV-2 revealed intracellular uptake of the virus for 24 h.p.i (A) , 48 h.p.i (B) and 72 h.p.i (C) . (D) plaque assay quantification from supernatant obtained from infected podocytes at 24, 48 and 72 h.p.i. (E) qPCR analysis of podocyte-specific genes revealed that both synaptopodin (SYNPO) and podocalyxin (PODXL) are significantly upregulated after infection with SARS-CoV-2 at MOI of 0.01, whereas SYNPO is significantly upregulated at multiple MOIs, and PODXL shows no significant changes with viral infection. (F) The expression of spike-associated genes (ACE2, BSG/CD147) and spike processing genes (TMPRSS2, CTSL) are significantly impacted by infection at MOIs of 0.01 and 0.1, respectively. (G) Human iPS cell-derived podocytes treated with SARS-CoV-2 (at MOI of 0.01) immunostain positive for Nucleocapsid protein (magenta), indicating successful infection with the virus. The cells were immunostained also for the podocyte marker Nephrin (green) and counterstained with DAPI (blue). Scale bar: 100 µm (H) Spike positive cells Nephrin and DAPI as nuclear counterstain in the infected podocytes. Scale bar: 100 µm. One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p

    Article Snippet: SARS-CoV-2 RNA was extracted from the supernatant or cell pellet of infected podocytes using the QIAamp viral RNA mini kit (Qiagen; 52904). qRT-PCR was performed with primers specific for target genes (see for the list of primers) using the Luna universal One-Step RT-qPCR kit (NEB; E3005).

    Techniques: Derivative Assay, Infection, Real-time Polymerase Chain Reaction, Plaque Assay, Expressing, Marker

    Transcriptomic and protein level analyses of spike-interacting factors in mature podocytes differentiated from human iPS cells. (A) Heatmap showing expression levels of spike associated genes from three biological human iPS cell-derived podocyte samples. SIGLEC9, Sialic acid-binding Ig-like lectin 9; CAPZA1, F-actin-capping protein subunit alpha-1; CLEC10A, C-type lectin domain family 10 member A; CD33, Myeloid cell surface antigen CD33; TMOD3, Tropomodulin-3; ACE2, Angiotensin Converting Enzyme 2; BSG/CD147, Basigin/CD147 molecule; CD209, CD209 Antigen; MYO6, Unconventional myosin-VI; PLS3, Plastin-3; LDHB, L-lactate dehydrogenase B chain; GNB2L1/RACK, Receptor of activated protein C kinase 1; SNRNP70, U1 small nuclear ribonucleoprotein 70 kDa; DOCK7, Dedicator of cytokinesis protein 7; RPS18, 40S ribosomal protein S18; CAPZB, F-actin-capping protein subunit beta; GOLGA7, Golgin subfamily A member 7; ZDHHC5, Palmitoyltransferase ZDHHC5; SIGLEC10, Sialic acid-binding Ig-like lectin 10; ACTR3, Actin-related protein 3; MYL6, Myosin light polypeptide 6; CORO1C, Coronin-1C; ARPC4, Actin-related protein 2/3 complex subunit 4; CCT6A, T-complex protein 1 subunit zeta; (B) qPCR quantification of nine of the human spike-associated gene from (A) including Transmembrane Serine Protease 2 (TMPRSS2) or cathepsin L (CTSL) in human iPS cell-derived podocytes, Calu-3, Caco-2, glomerular endothelial cells, and HEK 293T cells (normalized to Calu-3 groups). (C) Western blot analysis to evaluate protein expression of ACE2, BSG/CD147, CD209, TMPRSS2 and CTSL in the different cell type used; Calu-3, human iPS cell-derived podocytes, Caco-2, glomerular endothelial (gEndos) cells, and HEK 293T cells. (D) Immunocytochemistry analysis of ACE2, BSG/CD147, CD209, TMPRSS2 and CTSL expression in iPS cell-derived podocytes. Scale bar: 100 µm. One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: SARS-CoV-2 Employ BSG/CD147 and ACE2 Receptors to Directly Infect Human Induced Pluripotent Stem Cell-Derived Kidney Podocytes

    doi: 10.3389/fcell.2022.855340

    Figure Lengend Snippet: Transcriptomic and protein level analyses of spike-interacting factors in mature podocytes differentiated from human iPS cells. (A) Heatmap showing expression levels of spike associated genes from three biological human iPS cell-derived podocyte samples. SIGLEC9, Sialic acid-binding Ig-like lectin 9; CAPZA1, F-actin-capping protein subunit alpha-1; CLEC10A, C-type lectin domain family 10 member A; CD33, Myeloid cell surface antigen CD33; TMOD3, Tropomodulin-3; ACE2, Angiotensin Converting Enzyme 2; BSG/CD147, Basigin/CD147 molecule; CD209, CD209 Antigen; MYO6, Unconventional myosin-VI; PLS3, Plastin-3; LDHB, L-lactate dehydrogenase B chain; GNB2L1/RACK, Receptor of activated protein C kinase 1; SNRNP70, U1 small nuclear ribonucleoprotein 70 kDa; DOCK7, Dedicator of cytokinesis protein 7; RPS18, 40S ribosomal protein S18; CAPZB, F-actin-capping protein subunit beta; GOLGA7, Golgin subfamily A member 7; ZDHHC5, Palmitoyltransferase ZDHHC5; SIGLEC10, Sialic acid-binding Ig-like lectin 10; ACTR3, Actin-related protein 3; MYL6, Myosin light polypeptide 6; CORO1C, Coronin-1C; ARPC4, Actin-related protein 2/3 complex subunit 4; CCT6A, T-complex protein 1 subunit zeta; (B) qPCR quantification of nine of the human spike-associated gene from (A) including Transmembrane Serine Protease 2 (TMPRSS2) or cathepsin L (CTSL) in human iPS cell-derived podocytes, Calu-3, Caco-2, glomerular endothelial cells, and HEK 293T cells (normalized to Calu-3 groups). (C) Western blot analysis to evaluate protein expression of ACE2, BSG/CD147, CD209, TMPRSS2 and CTSL in the different cell type used; Calu-3, human iPS cell-derived podocytes, Caco-2, glomerular endothelial (gEndos) cells, and HEK 293T cells. (D) Immunocytochemistry analysis of ACE2, BSG/CD147, CD209, TMPRSS2 and CTSL expression in iPS cell-derived podocytes. Scale bar: 100 µm. One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p

    Article Snippet: SARS-CoV-2 RNA was extracted from the supernatant or cell pellet of infected podocytes using the QIAamp viral RNA mini kit (Qiagen; 52904). qRT-PCR was performed with primers specific for target genes (see for the list of primers) using the Luna universal One-Step RT-qPCR kit (NEB; E3005).

    Techniques: Expressing, Derivative Assay, Binding Assay, Real-time Polymerase Chain Reaction, Western Blot, Immunocytochemistry

    Antibody blocking reveal roles of ACE2 and BSG/CD147 receptors in viral uptake. (A) qPCR quantification of S-pseudotyped entry relative to antibody concentration (normalized to unblocked samples). Human iPS cell-derived podocytes were incubated with different dilutions of anti-ACE2, anti-BSG or both for an hour followed by infection with S-pseudotyped virus at MOI 0.02 for 60 h (B) qPCR quantification of Spike binding receptor genes (ACE2, BSG/CD147) and Spike processing factor genes (TMPRSS2, CTSL) at 5 µg/ml for both anti-ACE2 antibody blockage and anti-BSG antibody blockage showing significant changes in gene expression with optimal receptor blockage when compared to unblocked samples (normalized to mock groups). One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: SARS-CoV-2 Employ BSG/CD147 and ACE2 Receptors to Directly Infect Human Induced Pluripotent Stem Cell-Derived Kidney Podocytes

    doi: 10.3389/fcell.2022.855340

    Figure Lengend Snippet: Antibody blocking reveal roles of ACE2 and BSG/CD147 receptors in viral uptake. (A) qPCR quantification of S-pseudotyped entry relative to antibody concentration (normalized to unblocked samples). Human iPS cell-derived podocytes were incubated with different dilutions of anti-ACE2, anti-BSG or both for an hour followed by infection with S-pseudotyped virus at MOI 0.02 for 60 h (B) qPCR quantification of Spike binding receptor genes (ACE2, BSG/CD147) and Spike processing factor genes (TMPRSS2, CTSL) at 5 µg/ml for both anti-ACE2 antibody blockage and anti-BSG antibody blockage showing significant changes in gene expression with optimal receptor blockage when compared to unblocked samples (normalized to mock groups). One-way analysis of variance (ANOVA) with Sidak’s multiple comparison test was used to determine statistical significance. Only p values of 0.05 or lower were considered statistically significant ( p > 0.05 [ns, not significant], p

    Article Snippet: SARS-CoV-2 RNA was extracted from the supernatant or cell pellet of infected podocytes using the QIAamp viral RNA mini kit (Qiagen; 52904). qRT-PCR was performed with primers specific for target genes (see for the list of primers) using the Luna universal One-Step RT-qPCR kit (NEB; E3005).

    Techniques: Blocking Assay, Real-time Polymerase Chain Reaction, Concentration Assay, Derivative Assay, Incubation, Infection, Binding Assay, Expressing

    Knockdown (KD) of Qpr6 leads to the hypersensitivity of D. radiodurans to H 2 O 2 and chronic ionizing radiation (CIR). (A) Evaluation of Qpr6 level in R1, Qpr6 KD, and Qpr6 Com by RT-qPCR; (B) Survival of R1, Qpr6 KD, and Qpr6 Com under H 2 O 2 stress; (C) Growth of R1, Qpr6 KD, and Qpr6 Com under 0 and 57 Gy/h radiation ( γ -ray) for 6 days; (D) Survival rate of R1, Qpr6 KD, and Qpr6 Com under 10 kGy ionizing radiation ( β -ray). Error bars indicate standard errors of the means ( n = 3). ** p ≤ 0.01, and *** p ≤ 0.001 were considered as significant using the Student t -test, while ns indicates non-significant compared to R1 ( p > 0.05).

    Journal: Frontiers in Microbiology

    Article Title: Signal Recognition Particle RNA Contributes to Oxidative Stress Response in Deinococcus radiodurans by Modulating Catalase Localization

    doi: 10.3389/fmicb.2020.613571

    Figure Lengend Snippet: Knockdown (KD) of Qpr6 leads to the hypersensitivity of D. radiodurans to H 2 O 2 and chronic ionizing radiation (CIR). (A) Evaluation of Qpr6 level in R1, Qpr6 KD, and Qpr6 Com by RT-qPCR; (B) Survival of R1, Qpr6 KD, and Qpr6 Com under H 2 O 2 stress; (C) Growth of R1, Qpr6 KD, and Qpr6 Com under 0 and 57 Gy/h radiation ( γ -ray) for 6 days; (D) Survival rate of R1, Qpr6 KD, and Qpr6 Com under 10 kGy ionizing radiation ( β -ray). Error bars indicate standard errors of the means ( n = 3). ** p ≤ 0.01, and *** p ≤ 0.001 were considered as significant using the Student t -test, while ns indicates non-significant compared to R1 ( p > 0.05).

    Article Snippet: The RT-qPCR was performed using Luna Universal One-Step RT-qPCR Kit (New England Biolabs Inc.) on the ViiA7 instrument (Applied Biosystems).

    Techniques: Quantitative RT-PCR

    Mitoprotein-induced stress triggers the UPR ER . A , Fusion of DHFR to the N-terminus of Cytochrome b 2 generates a mitochondrial ‘clogger’ that jams the protein import machinery. B , The mitochondrial clogger b 2 -DHFR or cytosolic DHFR were expressed for 4.5 h. The precursor form of the mitochondrial proteins Mdj1 and Rip1 were detected by Western Blotting. C , Expression of b 2 -DHFR leads to attenuated growth. D , The mitoprotein-induced stress response encompasses an early transcriptional induction of chaperones and the proteasome and a downregulation of cytosolic ribosomes and OXPHOS components. E , Protein levels in clogger-expressing versus control cells after 18 h of induction were measured by quantitative mass spectrometry 28 . Highlighted are proteins which are reported targets of the UPR ER 21 . Data from n =3 independent biological replicates are shown. F, G , The cellular transcriptome and translatome after 4.5 h of clogger induction were measured by RNA-Seq ( n =4) 28 and ribosome profiling ( n =3), respectively. Shown are log 2 fold changes of b 2 -DHFR versus cytosolic DHFR. HAC1 transcripts are slightly reduced, but its translation is upregulated. H , Ribosome footprints along the HAC1 gene from cells expressing b 2 -DHFR or cytosolic DHFR for 4.5 h are shown. I , Levels of spliced HAC1 mRNA in cells expressing b 2 -DHFR or cytosolic DHFR were measured by RT-qPCR over time ( n =3).

    Journal: bioRxiv

    Article Title: The unfolded protein response of the endoplasmic reticulum supports mitochondrial biogenesis by buffering non-imported proteins

    doi: 10.1101/2021.05.19.444788

    Figure Lengend Snippet: Mitoprotein-induced stress triggers the UPR ER . A , Fusion of DHFR to the N-terminus of Cytochrome b 2 generates a mitochondrial ‘clogger’ that jams the protein import machinery. B , The mitochondrial clogger b 2 -DHFR or cytosolic DHFR were expressed for 4.5 h. The precursor form of the mitochondrial proteins Mdj1 and Rip1 were detected by Western Blotting. C , Expression of b 2 -DHFR leads to attenuated growth. D , The mitoprotein-induced stress response encompasses an early transcriptional induction of chaperones and the proteasome and a downregulation of cytosolic ribosomes and OXPHOS components. E , Protein levels in clogger-expressing versus control cells after 18 h of induction were measured by quantitative mass spectrometry 28 . Highlighted are proteins which are reported targets of the UPR ER 21 . Data from n =3 independent biological replicates are shown. F, G , The cellular transcriptome and translatome after 4.5 h of clogger induction were measured by RNA-Seq ( n =4) 28 and ribosome profiling ( n =3), respectively. Shown are log 2 fold changes of b 2 -DHFR versus cytosolic DHFR. HAC1 transcripts are slightly reduced, but its translation is upregulated. H , Ribosome footprints along the HAC1 gene from cells expressing b 2 -DHFR or cytosolic DHFR for 4.5 h are shown. I , Levels of spliced HAC1 mRNA in cells expressing b 2 -DHFR or cytosolic DHFR were measured by RT-qPCR over time ( n =3).

    Article Snippet: 100 ng total RNA per 20 µl reaction were analyzed using the Luna Universal Probe One-Step RT-qPCR Kit (NEB, # E3006) in technical triplicates. cDNA was generated by reverse transcription for 10 min at 55°C.

    Techniques: Western Blot, Expressing, Mass Spectrometry, RNA Sequencing Assay, Quantitative RT-PCR

    Detection of UPR ER induction with mass spectrometry and RT-qPCR. A , Protein levels in clogger-expressing versus control cells after different times of induction were measured by quantitative mass spectrometry 28 . Highlighted are proteins which are reported targets of the UPR ER 21 . Data from n =3 independent biological replicates are shown. The data for 18 h are the same as shown in Fig. 1E . B , The change in translational efficiency after 4.5 h clogger expression was calculated for all genes measured in both the RNA-seq 28 and Ribo-Seq on clogger-expressing cells by dividing the translatome fold change by the transcriptome fold change. C , Schematic depiction of the primer-probe combinations used to quantify total HAC1 as well as spliced HAC1 i mRNA levels via RT-qPCR. D , Wild type, Δ ire1 and Δ hac1 cells were grown in presence or absence of 1 µg/ml tunicamycin and HAC1 and HAC1 i levels were analyzed with the primer-probe assay shown in C. As expected, HAC1 i levels increased in wild type cells treated with tunicamyin, but no HAC1 i was detected in cells lacking HAC1 or IRE1 , confirming the specificity of the RT-qPCR assay.

    Journal: bioRxiv

    Article Title: The unfolded protein response of the endoplasmic reticulum supports mitochondrial biogenesis by buffering non-imported proteins

    doi: 10.1101/2021.05.19.444788

    Figure Lengend Snippet: Detection of UPR ER induction with mass spectrometry and RT-qPCR. A , Protein levels in clogger-expressing versus control cells after different times of induction were measured by quantitative mass spectrometry 28 . Highlighted are proteins which are reported targets of the UPR ER 21 . Data from n =3 independent biological replicates are shown. The data for 18 h are the same as shown in Fig. 1E . B , The change in translational efficiency after 4.5 h clogger expression was calculated for all genes measured in both the RNA-seq 28 and Ribo-Seq on clogger-expressing cells by dividing the translatome fold change by the transcriptome fold change. C , Schematic depiction of the primer-probe combinations used to quantify total HAC1 as well as spliced HAC1 i mRNA levels via RT-qPCR. D , Wild type, Δ ire1 and Δ hac1 cells were grown in presence or absence of 1 µg/ml tunicamycin and HAC1 and HAC1 i levels were analyzed with the primer-probe assay shown in C. As expected, HAC1 i levels increased in wild type cells treated with tunicamyin, but no HAC1 i was detected in cells lacking HAC1 or IRE1 , confirming the specificity of the RT-qPCR assay.

    Article Snippet: 100 ng total RNA per 20 µl reaction were analyzed using the Luna Universal Probe One-Step RT-qPCR Kit (NEB, # E3006) in technical triplicates. cDNA was generated by reverse transcription for 10 min at 55°C.

    Techniques: Mass Spectrometry, Quantitative RT-PCR, Expressing, RNA Sequencing Assay