luna universal qpcr master mix  (New England Biolabs)


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    Structured Review

    New England Biolabs luna universal qpcr master mix
    Generation of TUBL-deficient mice. (A) Schematic representation of the WT TINCR allele, the single-stranded oligodeoxynucleotide (ssODN), and the mutant allele after homologous recombination. Exons are denoted by numbered boxes. The single guide RNA (sgRNA) for the CRISPR-Cas9 system and its protospacer adjacent motif (PAM) are indicated by contiguous black and red underlines, respectively. The TUBL ORF is represented by the gray shading in the box corresponding to exon 1 of TINCR . (B) Predicted secondary structure and minimal free energy for WT TINCR and the mutant form generated by the CRISPR-Cas9 system for establishment of Tubl −/− mice. The triangle indicates the 5’ end of the transcript. (C) <t>PCR</t> analysis of genomic DNA from the tail of mice of the indicated genotypes. The PCR products were digested with EcoRI before electrophoresis. (D) <t>RT-qPCR</t> analysis of TINCR in the epidermis of Tubl +/+ and Tubl −/− mice. Data are means ± SD (n = 3 independent experiments). ***p
    Luna Universal Qpcr Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A ubiquitin-like protein encoded by the “noncoding” RNA TINCR promotes keratinocyte proliferation and wound healing"

    Article Title: A ubiquitin-like protein encoded by the “noncoding” RNA TINCR promotes keratinocyte proliferation and wound healing

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1009686

    Generation of TUBL-deficient mice. (A) Schematic representation of the WT TINCR allele, the single-stranded oligodeoxynucleotide (ssODN), and the mutant allele after homologous recombination. Exons are denoted by numbered boxes. The single guide RNA (sgRNA) for the CRISPR-Cas9 system and its protospacer adjacent motif (PAM) are indicated by contiguous black and red underlines, respectively. The TUBL ORF is represented by the gray shading in the box corresponding to exon 1 of TINCR . (B) Predicted secondary structure and minimal free energy for WT TINCR and the mutant form generated by the CRISPR-Cas9 system for establishment of Tubl −/− mice. The triangle indicates the 5’ end of the transcript. (C) PCR analysis of genomic DNA from the tail of mice of the indicated genotypes. The PCR products were digested with EcoRI before electrophoresis. (D) RT-qPCR analysis of TINCR in the epidermis of Tubl +/+ and Tubl −/− mice. Data are means ± SD (n = 3 independent experiments). ***p
    Figure Legend Snippet: Generation of TUBL-deficient mice. (A) Schematic representation of the WT TINCR allele, the single-stranded oligodeoxynucleotide (ssODN), and the mutant allele after homologous recombination. Exons are denoted by numbered boxes. The single guide RNA (sgRNA) for the CRISPR-Cas9 system and its protospacer adjacent motif (PAM) are indicated by contiguous black and red underlines, respectively. The TUBL ORF is represented by the gray shading in the box corresponding to exon 1 of TINCR . (B) Predicted secondary structure and minimal free energy for WT TINCR and the mutant form generated by the CRISPR-Cas9 system for establishment of Tubl −/− mice. The triangle indicates the 5’ end of the transcript. (C) PCR analysis of genomic DNA from the tail of mice of the indicated genotypes. The PCR products were digested with EcoRI before electrophoresis. (D) RT-qPCR analysis of TINCR in the epidermis of Tubl +/+ and Tubl −/− mice. Data are means ± SD (n = 3 independent experiments). ***p

    Techniques Used: Mouse Assay, Mutagenesis, Homologous Recombination, CRISPR, Generated, Polymerase Chain Reaction, Electrophoresis, Quantitative RT-PCR

    2) Product Images from "MEOX2 Regulates the Growth and Survival of Glioblastoma Stem Cells by Modulating Genes of the Glycolytic Pathway and Response to Hypoxia"

    Article Title: MEOX2 Regulates the Growth and Survival of Glioblastoma Stem Cells by Modulating Genes of the Glycolytic Pathway and Response to Hypoxia

    Journal: Cancers

    doi: 10.3390/cancers14092304

    MEOX2 depletion inhibits the sphere-forming and the growth ability of glioblastoma stem cells BT273 and BT379. ( a , b ) MEOX2 qRT-PCR analysis of BT273 ( a ) or BT379 ( b ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. The values were reported in relation to cells transduced with ctrl vector set as = 1 and normalized to PPP2CA mRNA expression ( n = 3; mean ± SD). ( c , d ) MEOX2 Western blot analysis of BT273 ( c ) or BT379 ( d ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. For BT273 and BT379, α-tubulin and β-actin were used as the internal loading controls, respectively. Representative images are shown. The bottom histograms show the quantification of MEOX2 in relation to α-tubulin and β-actin. ( n = 3; mean ± SD). ( e , f ) Sphere-forming assay of BT273 ( e ) and BT379 ( f ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. Histograms show the percentage of cells capable of re-forming a neurosphere seven days after dissociation ( n = 3; mean ± SD). Representative micrographs are shown. ( g , h ) Growth curves of BT273 ( g ) and BT379 ( h ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. ( n = 3; mean ± SD). Differences between two groups were assessed using unpaired Student’s t -test (two-tailed). Significance was defined as * p
    Figure Legend Snippet: MEOX2 depletion inhibits the sphere-forming and the growth ability of glioblastoma stem cells BT273 and BT379. ( a , b ) MEOX2 qRT-PCR analysis of BT273 ( a ) or BT379 ( b ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. The values were reported in relation to cells transduced with ctrl vector set as = 1 and normalized to PPP2CA mRNA expression ( n = 3; mean ± SD). ( c , d ) MEOX2 Western blot analysis of BT273 ( c ) or BT379 ( d ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. For BT273 and BT379, α-tubulin and β-actin were used as the internal loading controls, respectively. Representative images are shown. The bottom histograms show the quantification of MEOX2 in relation to α-tubulin and β-actin. ( n = 3; mean ± SD). ( e , f ) Sphere-forming assay of BT273 ( e ) and BT379 ( f ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. Histograms show the percentage of cells capable of re-forming a neurosphere seven days after dissociation ( n = 3; mean ± SD). Representative micrographs are shown. ( g , h ) Growth curves of BT273 ( g ) and BT379 ( h ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. ( n = 3; mean ± SD). Differences between two groups were assessed using unpaired Student’s t -test (two-tailed). Significance was defined as * p

    Techniques Used: Quantitative RT-PCR, Transduction, Construct, Plasmid Preparation, Expressing, Western Blot, Two Tailed Test

    3) Product Images from "Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function"

    Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

    Journal: bioRxiv

    doi: 10.1101/2022.05.20.492787

    a) Distribution of annotated single hits over MEG3 gene, with statistically filtered EZH2-FLASH reads from two biological replicates in HUVECs. b) The occupancy of EZH2 hits over MEG3 features. Total reads per feature are given with exons being mostly occupies vs introns. c) Proportion of overlapping features over MEG3. The occupancy of EZH2 over each MEG3 exon is shown for two constitutively expressed transcripts. For both given transcripts there is high occupancy of exon 3. d) RNA immunoprecipitation (RIP) for EZH2 and H3K27me3 (repressive chromatin) followed by qPCR analysis. RIP-purified RNA from UV crosslinked HUVECs was used to prepare cDNA for qPCR analysis with primers against MEG3 (exon 3 region). Primers against U1snRNA gene serves as a negative control. Side diagram of EHZ2-MEG3 interacting region is charted as per FLASH hits and sequence. e) Distribution of EZH2 hybrids hits over MEG3 gene. Intermolecular MEG3-RNA interactions found in chimeras are captured by EZH2-FLASH-seq. Hits represent MEG3:MEG3 hybrids (black). IgG hybrids are plotted but are
    Figure Legend Snippet: a) Distribution of annotated single hits over MEG3 gene, with statistically filtered EZH2-FLASH reads from two biological replicates in HUVECs. b) The occupancy of EZH2 hits over MEG3 features. Total reads per feature are given with exons being mostly occupies vs introns. c) Proportion of overlapping features over MEG3. The occupancy of EZH2 over each MEG3 exon is shown for two constitutively expressed transcripts. For both given transcripts there is high occupancy of exon 3. d) RNA immunoprecipitation (RIP) for EZH2 and H3K27me3 (repressive chromatin) followed by qPCR analysis. RIP-purified RNA from UV crosslinked HUVECs was used to prepare cDNA for qPCR analysis with primers against MEG3 (exon 3 region). Primers against U1snRNA gene serves as a negative control. Side diagram of EHZ2-MEG3 interacting region is charted as per FLASH hits and sequence. e) Distribution of EZH2 hybrids hits over MEG3 gene. Intermolecular MEG3-RNA interactions found in chimeras are captured by EZH2-FLASH-seq. Hits represent MEG3:MEG3 hybrids (black). IgG hybrids are plotted but are

    Techniques Used: Immunoprecipitation, Real-time Polymerase Chain Reaction, Purification, Negative Control, Sequencing

    a. Venn diagram showing the intersection between statistically filtered FLASH data from two biological replicates of our MEG3-ChIRP-seq-data (green), de novo hg38 analysed GEO RNA-seq data from siEZH2 deficient HUVECs (GSE71164, blue), and EZH2 ChIP-seq following MEG3 KD (yellow) and FLASH-seq transcriptome following EZH2 IP (pink). b. Correlation between gene expression levels and FLASH signal. Gray, expressed RefSeq genes with reproducible FLASH signal consistently detected in RNA-seq. Blue, genes with the highest RNA-seq signals and no reproducible FLASH signal belonging to integrin cell surface interaction pathway. Red , expressed ITGA4 gene, and green, ITGB1 gene, without reproducible FLASH signals. Data are from two biological replicates of each EZH2 FLASH sample and three biological replicates of EZH2 RNA-seq samples (Scr vs. siEZH2, GSE71164). c. Genomic tracks showing ChIRP-seq signal (MEG3 Odd, Even and LacZ) in HUVECs over ITGA4 gene only. The MEG3 binding site is located upstream of the ITGA4 gene in the promoter region, and it overlaps with the H3K27me3 signal and EZH2; as well as downstream within the ITGA4 gene body, where it overlaps with within the EZH2 signal in the intronic region of the gene. d. MEG3-ChIRP followed by qPCR, analysis of MEG3 binding region on ITGA4 in HUVECs. The crosslinked cell lysates were incubated with combined biotinylated probes against MEG3 lncRNA and the binding complexes recovered by magnetic streptavidin-conjugated beads. The qPCR was performed to detect the enrichment of specific region that associated with MEG3, peaks were related to input control and compared vs. the non-biotynilated control. e. ChIP-QPCR enrichment for EZH2 and H3K27me3 over ITGA4 promoter region in HUVECs depleted of MEG3 vs. Control.
    Figure Legend Snippet: a. Venn diagram showing the intersection between statistically filtered FLASH data from two biological replicates of our MEG3-ChIRP-seq-data (green), de novo hg38 analysed GEO RNA-seq data from siEZH2 deficient HUVECs (GSE71164, blue), and EZH2 ChIP-seq following MEG3 KD (yellow) and FLASH-seq transcriptome following EZH2 IP (pink). b. Correlation between gene expression levels and FLASH signal. Gray, expressed RefSeq genes with reproducible FLASH signal consistently detected in RNA-seq. Blue, genes with the highest RNA-seq signals and no reproducible FLASH signal belonging to integrin cell surface interaction pathway. Red , expressed ITGA4 gene, and green, ITGB1 gene, without reproducible FLASH signals. Data are from two biological replicates of each EZH2 FLASH sample and three biological replicates of EZH2 RNA-seq samples (Scr vs. siEZH2, GSE71164). c. Genomic tracks showing ChIRP-seq signal (MEG3 Odd, Even and LacZ) in HUVECs over ITGA4 gene only. The MEG3 binding site is located upstream of the ITGA4 gene in the promoter region, and it overlaps with the H3K27me3 signal and EZH2; as well as downstream within the ITGA4 gene body, where it overlaps with within the EZH2 signal in the intronic region of the gene. d. MEG3-ChIRP followed by qPCR, analysis of MEG3 binding region on ITGA4 in HUVECs. The crosslinked cell lysates were incubated with combined biotinylated probes against MEG3 lncRNA and the binding complexes recovered by magnetic streptavidin-conjugated beads. The qPCR was performed to detect the enrichment of specific region that associated with MEG3, peaks were related to input control and compared vs. the non-biotynilated control. e. ChIP-QPCR enrichment for EZH2 and H3K27me3 over ITGA4 promoter region in HUVECs depleted of MEG3 vs. Control.

    Techniques Used: RNA Sequencing Assay, Chromatin Immunoprecipitation, Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Incubation

    a. ChIP signal enrichment vs . 1% input for EZH2 and H3K27me3 mark over ITGA4 promoter regions in HUVECs treated with A-395 (5µM, 24h) inhibitor of PRC2 vs. Control (DMSO). The expression was measured using two sets of primers against the same promoter region of ITGA4. Representative graphs are average of three qPCR datasets ± SEM. b. ITGA4 expression in the presence of A-395 vs . DMSO control, N=6 independent experiments compared using t -test. c. Measuring the expression levels of ITGA4 upon depletion of MEG3 using LNA GapmeRs (10nM, 48h), data is mean of N=5 independent experiments (biological replicates). d. Representative image of immunofluorescence staining for ITGA4 protein levels in ECs treated with A-395 vs . DMSO, or upon MEG3 depletion like in b . e. Intra-cellular localisation of MEG3 (chromatin associated lncRNA) between different cellular compartments in HUVECs treated with A-395 vs. DMSO, whereby the distribution of MEG3 has shifted upon PRC2 inhibition with A-395; from the nucleus (where it was highly chromatin bound) into the cytoplasm. Representative bars were compared by t-test and on-way Anova. f. MEG3-ChIRP followed by qPCR, N =3, analysis of MEG3 binding over ITGA4 promoter region in HUVECs treated with A-395 (5µM, 24h) vs. DMSO. MEG3-ChIRP HUVEC lysates treated with A-395 resulted in reduced engagement of MEG3 with ITGA4 site compared with either DMSO control or ChIRP with non-biotinylated probes. The non-biotin probes served as a negative control, and we detected the background level
    Figure Legend Snippet: a. ChIP signal enrichment vs . 1% input for EZH2 and H3K27me3 mark over ITGA4 promoter regions in HUVECs treated with A-395 (5µM, 24h) inhibitor of PRC2 vs. Control (DMSO). The expression was measured using two sets of primers against the same promoter region of ITGA4. Representative graphs are average of three qPCR datasets ± SEM. b. ITGA4 expression in the presence of A-395 vs . DMSO control, N=6 independent experiments compared using t -test. c. Measuring the expression levels of ITGA4 upon depletion of MEG3 using LNA GapmeRs (10nM, 48h), data is mean of N=5 independent experiments (biological replicates). d. Representative image of immunofluorescence staining for ITGA4 protein levels in ECs treated with A-395 vs . DMSO, or upon MEG3 depletion like in b . e. Intra-cellular localisation of MEG3 (chromatin associated lncRNA) between different cellular compartments in HUVECs treated with A-395 vs. DMSO, whereby the distribution of MEG3 has shifted upon PRC2 inhibition with A-395; from the nucleus (where it was highly chromatin bound) into the cytoplasm. Representative bars were compared by t-test and on-way Anova. f. MEG3-ChIRP followed by qPCR, N =3, analysis of MEG3 binding over ITGA4 promoter region in HUVECs treated with A-395 (5µM, 24h) vs. DMSO. MEG3-ChIRP HUVEC lysates treated with A-395 resulted in reduced engagement of MEG3 with ITGA4 site compared with either DMSO control or ChIRP with non-biotinylated probes. The non-biotin probes served as a negative control, and we detected the background level

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Inhibition, Binding Assay, Negative Control

    a) Overview of the design of probes against MEG3 gene that were divided in probe Set1 and Set 2. The biotynilated probes were of 20 nucleotides and were spaced out 200 nucleotides apart down the gene length. b) Validation of MEG3 probes specifically binding MEG3 gene, by ChIRP-qPCR in HUVECs. Pull down with probe set 1 or set 2 retrieved 100% and 40% RNA, respectively. GAPDH primers were used as control and MEG3-associated samples did not amplify. c) Computational analysis pipeline for ChIRP-seq outlining data processing. The peak coverage was within the 100bp window. d) MEG3-ChIRP peaks associated with EZH2 gene as precipitated using both sets of probes (set 1 and 2). e) Enrichment of MEG3 signal by ChIRP-qpcr versus negative control (Background) at named promoter regions. MEG3 binding to genomic loci as validate by ChIRP-qPCR in HUVECs. Pull downs were performed with joined Odd and Even probes. Value 1 is a background level, defined by enrichment to LacZ negative probes in ChIRP. Control primers were designed for positive ChIRP peaks and used as a positive control and for regions deprived of MEG3-ChIRP reads as a negative control .
    Figure Legend Snippet: a) Overview of the design of probes against MEG3 gene that were divided in probe Set1 and Set 2. The biotynilated probes were of 20 nucleotides and were spaced out 200 nucleotides apart down the gene length. b) Validation of MEG3 probes specifically binding MEG3 gene, by ChIRP-qPCR in HUVECs. Pull down with probe set 1 or set 2 retrieved 100% and 40% RNA, respectively. GAPDH primers were used as control and MEG3-associated samples did not amplify. c) Computational analysis pipeline for ChIRP-seq outlining data processing. The peak coverage was within the 100bp window. d) MEG3-ChIRP peaks associated with EZH2 gene as precipitated using both sets of probes (set 1 and 2). e) Enrichment of MEG3 signal by ChIRP-qpcr versus negative control (Background) at named promoter regions. MEG3 binding to genomic loci as validate by ChIRP-qPCR in HUVECs. Pull downs were performed with joined Odd and Even probes. Value 1 is a background level, defined by enrichment to LacZ negative probes in ChIRP. Control primers were designed for positive ChIRP peaks and used as a positive control and for regions deprived of MEG3-ChIRP reads as a negative control .

    Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Negative Control, Positive Control

    4) Product Images from "Antiproliferative and Pro-Apoptotic Effects of a Phenolic-Rich Extract from Lycium barbarum Fruits on Human Papillomavirus (HPV) 16-Positive Head Cancer Cell Lines"

    Article Title: Antiproliferative and Pro-Apoptotic Effects of a Phenolic-Rich Extract from Lycium barbarum Fruits on Human Papillomavirus (HPV) 16-Positive Head Cancer Cell Lines

    Journal: Molecules

    doi: 10.3390/molecules27113568

    mRNA expression levels in SCC090 cells treated with extracts from L. barbarum and C. sinensis for 48 h at 1.0, 10, and 100 μg/mL. The mRNA levels were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR): ( A ) E6 and E7, ( B ) p53. Results are expressed as the mean of three independent triplicate trials ( n = 3) ± standard error of the mean (SEM). The effect of the extract was compared concerning the treatments: * control (–) (untreated cells) and ** control (+) cisplatin ( p
    Figure Legend Snippet: mRNA expression levels in SCC090 cells treated with extracts from L. barbarum and C. sinensis for 48 h at 1.0, 10, and 100 μg/mL. The mRNA levels were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR): ( A ) E6 and E7, ( B ) p53. Results are expressed as the mean of three independent triplicate trials ( n = 3) ± standard error of the mean (SEM). The effect of the extract was compared concerning the treatments: * control (–) (untreated cells) and ** control (+) cisplatin ( p

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

    5) Product Images from "AMPK Enhances Transcription of Selected Nrf2 Target Genes via Negative Regulation of Bach1"

    Article Title: AMPK Enhances Transcription of Selected Nrf2 Target Genes via Negative Regulation of Bach1

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2020.00628

    AMPK regulates Bach1 at the transcriptional level. (A) Wt and AMPK−/− cells were treated with 10 μM cycloheximide for the indicated periods of time. Nuclear and cytosolic fractions were then probed for Bach1 and actin. Representative blots of three performed experiments are depicted together with compiled densitometric data showing the decay of Bach1 over time. (B) Wt and AMPK−/− cells were treated with DMSO (D, 0.1%) or Sfn (5 μM) for 4 h before bach1 mRNA levels were determined by qPCR ( hprt1 as reference gene) ( n = 3, mean + SD; two-way ANOVA, Tuckey post hoc test; * P ≤ 0.05).
    Figure Legend Snippet: AMPK regulates Bach1 at the transcriptional level. (A) Wt and AMPK−/− cells were treated with 10 μM cycloheximide for the indicated periods of time. Nuclear and cytosolic fractions were then probed for Bach1 and actin. Representative blots of three performed experiments are depicted together with compiled densitometric data showing the decay of Bach1 over time. (B) Wt and AMPK−/− cells were treated with DMSO (D, 0.1%) or Sfn (5 μM) for 4 h before bach1 mRNA levels were determined by qPCR ( hprt1 as reference gene) ( n = 3, mean + SD; two-way ANOVA, Tuckey post hoc test; * P ≤ 0.05).

    Techniques Used: Real-time Polymerase Chain Reaction

    AMPK-mediated boosted transcription of selected Nrf2 target genes is confirmed by qPCR analysis. (A) Wt and AMPK −/− MEFs were treated with DMSO (0.1%) or sulforaphane (Sfn, 5 μM) for 4 h before RNA was isolated, reversely transcribed and subjected to qPCR analysis for hmox1, akr1c14, nqo1, gclc, txnrd1 and gsta4 as indicated ( hprt1 as reference gene). (B) Experimental set up as in panel (A) , except that cells were (co)-treated with the AMPK inhibitor SBI0206965 (SBI, 30 μM) as indicated. (A,B) Bar graphs present the mean + 95% CI ( n = 3–4; * P ≤ 0.05, two-way ANOVA, Tuckey post-hoc test; ns: not significant).
    Figure Legend Snippet: AMPK-mediated boosted transcription of selected Nrf2 target genes is confirmed by qPCR analysis. (A) Wt and AMPK −/− MEFs were treated with DMSO (0.1%) or sulforaphane (Sfn, 5 μM) for 4 h before RNA was isolated, reversely transcribed and subjected to qPCR analysis for hmox1, akr1c14, nqo1, gclc, txnrd1 and gsta4 as indicated ( hprt1 as reference gene). (B) Experimental set up as in panel (A) , except that cells were (co)-treated with the AMPK inhibitor SBI0206965 (SBI, 30 μM) as indicated. (A,B) Bar graphs present the mean + 95% CI ( n = 3–4; * P ≤ 0.05, two-way ANOVA, Tuckey post-hoc test; ns: not significant).

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation

    Wt and AMPK−/− cells show distinct relative ratios of Bach1 and Nrf2 at ARE sites. Wt and AMPK−/− cells were treated with Sfn (5 μM) or 0.05% DMSO for 3 h. ChIPs were performed as described in section “Materials and Methods” using antibodies specific for Nrf2 or Bach1 and IgG (negative control), before isolating DNA for qPCR analysis of the indicated ARE-sites within hmox1 , nqo1 and gsta4 genes. The bar graphs depict compiled mean fold enrichment + 95% CI ( n = 3) of Nrf2 (green) and Bach1 (red) at the respective sites, calculated in reference to IgG (white) immunoprecipitates ( *P ≤ 0.05; ANOVA, Tuckey post-hoc test).
    Figure Legend Snippet: Wt and AMPK−/− cells show distinct relative ratios of Bach1 and Nrf2 at ARE sites. Wt and AMPK−/− cells were treated with Sfn (5 μM) or 0.05% DMSO for 3 h. ChIPs were performed as described in section “Materials and Methods” using antibodies specific for Nrf2 or Bach1 and IgG (negative control), before isolating DNA for qPCR analysis of the indicated ARE-sites within hmox1 , nqo1 and gsta4 genes. The bar graphs depict compiled mean fold enrichment + 95% CI ( n = 3) of Nrf2 (green) and Bach1 (red) at the respective sites, calculated in reference to IgG (white) immunoprecipitates ( *P ≤ 0.05; ANOVA, Tuckey post-hoc test).

    Techniques Used: Negative Control, Real-time Polymerase Chain Reaction

    Wt and AMPK −/− cells hardly differ in the chromatin opening at selected ARE sites. Cells were treated with DMSO (0.05%) or Sfn (5 μM) as indicated for 3 h. After crosslinking and chromatin shearing, free and histone-bound DNA were subjected to FAIRE-qPCR analysis for selected sites as described in detail in section “Materials and Methods.” As positive control a regulatory region within actin gene was used (mean chromatin opening of 1.0; green dotted line), and heterochromatin served as negative control for little chromatin accessibility (red dotted line); hmox-1-1,2,3: ARE sites within proximal promoter or within distal enhancer regions E1 and E2; nqo1, gsta4: ARE sites within proximal promoters. Bar graph depicts compiled data of three independent experiments. (mean + 95% CI; n = 3; * P ≤ 0.05, two-way ANOVA, Tuckey post-hoc test; ns: not significant).
    Figure Legend Snippet: Wt and AMPK −/− cells hardly differ in the chromatin opening at selected ARE sites. Cells were treated with DMSO (0.05%) or Sfn (5 μM) as indicated for 3 h. After crosslinking and chromatin shearing, free and histone-bound DNA were subjected to FAIRE-qPCR analysis for selected sites as described in detail in section “Materials and Methods.” As positive control a regulatory region within actin gene was used (mean chromatin opening of 1.0; green dotted line), and heterochromatin served as negative control for little chromatin accessibility (red dotted line); hmox-1-1,2,3: ARE sites within proximal promoter or within distal enhancer regions E1 and E2; nqo1, gsta4: ARE sites within proximal promoters. Bar graph depicts compiled data of three independent experiments. (mean + 95% CI; n = 3; * P ≤ 0.05, two-way ANOVA, Tuckey post-hoc test; ns: not significant).

    Techniques Used: Real-time Polymerase Chain Reaction, Positive Control, Negative Control

    Inhibition of Bach1 by hemin annihilates the distinct Nrf2 target gene expression between wt and AMPK−/− cells. Wt and AMPK −/− MEFs were treated with DMSO (0.1%), Sfn (5 μM) or hemin (Hem, 20 μM) as indicated. After for 4 h RNA was isolated, reversely transcribed and subjected to qPCR analysis for hmox1 , gclc, nqo1, txnrd 1 or hprt as reference gene ( n = 3, mean + SD; two-way ANOVA, Tuckey post hoc test; * P ≤ 0.05).
    Figure Legend Snippet: Inhibition of Bach1 by hemin annihilates the distinct Nrf2 target gene expression between wt and AMPK−/− cells. Wt and AMPK −/− MEFs were treated with DMSO (0.1%), Sfn (5 μM) or hemin (Hem, 20 μM) as indicated. After for 4 h RNA was isolated, reversely transcribed and subjected to qPCR analysis for hmox1 , gclc, nqo1, txnrd 1 or hprt as reference gene ( n = 3, mean + SD; two-way ANOVA, Tuckey post hoc test; * P ≤ 0.05).

    Techniques Used: Inhibition, Expressing, Isolation, Real-time Polymerase Chain Reaction

    6) Product Images from "ATR protects centromere identity by promoting DAXX association with PML nuclear bodies"

    Article Title: ATR protects centromere identity by promoting DAXX association with PML nuclear bodies

    Journal: bioRxiv

    doi: 10.1101/2022.09.26.509554

    ATR kinase activity protects CENP-A occupancy at interphase centromeres. (A-B) ATR inhibition reduces maximum nuclear CENP-A intensity . (A) Quantification of CENP-A intensity in Asynchronous U2OS cells that were untreated (CONT) or treated for 1 hour with 10μM VE-821 or 10μM AZ20 (ATRi). ( > 180 cells per condition; n=3 biological replicates). Error bars represent mean ±SD. (B) Representative images of cells stained for CENP-A (green) and DNA (DAPI; blue). (C) ATR inhibition reduces CENP-A occupancy at interphase centromeres . Quantitative PCR of CENP-A chromatin immunoprecipitation in asynchronous U2OS cells treated as in (A). Centromeres were amplified using primers for the core α-satellite sequences of chromosomes 1, 5 and 19 (αSat-1). Error bars represent mean ±SEM. (D-E) CENP-A loss with ATR inhibition is rapid . (D) Asynchronous U2OS cells stably expressing CENP-A-mCherry were treated as in (A). The intensity of the 3 brightest CENP-A foci were quantified and averaged per cell per time point and normalized to 100% at time = 0 minutes. (10 cells per condition, n = 1 biological replicate). Error bars represent mean ±SEM. (E) Representative images of U2OS CENP-A-mCherry cells quantified in (D). Inset depicts the 3 brightest foci quantified for that cell. (F-G) ATR inhibition induces CENP-A loss in HeLa and RPE-1 cells . Quantification of maximum nuclear CENP-A intensity in asynchronous HeLa (F) and RPE-1 (G) cells treated as in (A). ( > 130 HeLa cells per condition and > 100 RPE-1 cells per condition; n = 3 biological replicates). Error bars represent mean ±SD. *p ≤ 0.05, **p ≤ 0.01, two-tailed t-test of replicate averages. Scale bars for all panels = 5 μm.
    Figure Legend Snippet: ATR kinase activity protects CENP-A occupancy at interphase centromeres. (A-B) ATR inhibition reduces maximum nuclear CENP-A intensity . (A) Quantification of CENP-A intensity in Asynchronous U2OS cells that were untreated (CONT) or treated for 1 hour with 10μM VE-821 or 10μM AZ20 (ATRi). ( > 180 cells per condition; n=3 biological replicates). Error bars represent mean ±SD. (B) Representative images of cells stained for CENP-A (green) and DNA (DAPI; blue). (C) ATR inhibition reduces CENP-A occupancy at interphase centromeres . Quantitative PCR of CENP-A chromatin immunoprecipitation in asynchronous U2OS cells treated as in (A). Centromeres were amplified using primers for the core α-satellite sequences of chromosomes 1, 5 and 19 (αSat-1). Error bars represent mean ±SEM. (D-E) CENP-A loss with ATR inhibition is rapid . (D) Asynchronous U2OS cells stably expressing CENP-A-mCherry were treated as in (A). The intensity of the 3 brightest CENP-A foci were quantified and averaged per cell per time point and normalized to 100% at time = 0 minutes. (10 cells per condition, n = 1 biological replicate). Error bars represent mean ±SEM. (E) Representative images of U2OS CENP-A-mCherry cells quantified in (D). Inset depicts the 3 brightest foci quantified for that cell. (F-G) ATR inhibition induces CENP-A loss in HeLa and RPE-1 cells . Quantification of maximum nuclear CENP-A intensity in asynchronous HeLa (F) and RPE-1 (G) cells treated as in (A). ( > 130 HeLa cells per condition and > 100 RPE-1 cells per condition; n = 3 biological replicates). Error bars represent mean ±SD. *p ≤ 0.05, **p ≤ 0.01, two-tailed t-test of replicate averages. Scale bars for all panels = 5 μm.

    Techniques Used: Activity Assay, Inhibition, Staining, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Amplification, Stable Transfection, Expressing, Two Tailed Test

    7) Product Images from "Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses"

    Article Title: Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

    Journal: bioRxiv

    doi: 10.1101/2021.06.21.449162

    HMC3 cells respond to the recombinant cytokines IFNγ and IL1β and endogenously express β2AR. a: Quantitative reverse transcription PCR (RT-qPCR) of HMC3 cells exposed to recombinant interferon γ (IFNγ) and interleukin 1β (IL1β). Expression values of interleukin 6 (IL6), tumor necrosis factor (TNF), and IL1β shown as fold change compared to untreated cells (dashed line). Error bars: standard error of the mean of eight repetitions. One-sample T-test: p***
    Figure Legend Snippet: HMC3 cells respond to the recombinant cytokines IFNγ and IL1β and endogenously express β2AR. a: Quantitative reverse transcription PCR (RT-qPCR) of HMC3 cells exposed to recombinant interferon γ (IFNγ) and interleukin 1β (IL1β). Expression values of interleukin 6 (IL6), tumor necrosis factor (TNF), and IL1β shown as fold change compared to untreated cells (dashed line). Error bars: standard error of the mean of eight repetitions. One-sample T-test: p***

    Techniques Used: Recombinant, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    DREADD-based chimeras modulate inflammatory gene expression. a-f: Quantitative reverse transcription PCR (RT-qPCR) for interleukin 6 (IL6, top row), tumor necrosis factor (TNF, middle row), and interleukin 1β (IL1β, bottom row). Different HMC3 cell lines simultaneously treated with recombinant interferon γ (IFNγ) and interleukin 1β, and combinations of levalbuterol (LB, a ), CNO ( b-e ), or forskolin (FSK, f ). Graphs show fold changes compared to untreated cells with the IFNγ/IL1β treatment set to 100% within each repetition (dashed line). Dotted line: level of untreated controls. Lines connecting dots: dependent samples within experimental repetitions. Error bars: standard error of the mean of three to four repetitions. Linear regression analysis: p***
    Figure Legend Snippet: DREADD-based chimeras modulate inflammatory gene expression. a-f: Quantitative reverse transcription PCR (RT-qPCR) for interleukin 6 (IL6, top row), tumor necrosis factor (TNF, middle row), and interleukin 1β (IL1β, bottom row). Different HMC3 cell lines simultaneously treated with recombinant interferon γ (IFNγ) and interleukin 1β, and combinations of levalbuterol (LB, a ), CNO ( b-e ), or forskolin (FSK, f ). Graphs show fold changes compared to untreated cells with the IFNγ/IL1β treatment set to 100% within each repetition (dashed line). Dotted line: level of untreated controls. Lines connecting dots: dependent samples within experimental repetitions. Error bars: standard error of the mean of three to four repetitions. Linear regression analysis: p***

    Techniques Used: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Recombinant

    8) Product Images from "Drosophila STING protein has a role in lipid metabolism"

    Article Title: Drosophila STING protein has a role in lipid metabolism

    Journal: eLife

    doi: 10.7554/eLife.67358

    GFP-tagged Drosophila STING expression in different tissues. ( A ) Transcription of dSTING in wild type ( w 1118 ), mutant ( dSTINGΔ ), or ‘rescued’ ( dSTINGΔ;GFP-dSTING ) flies was measured by RT-qPCR. The bars indicate dSTING relative expression levels. Values were normalized to the wild type ( w 1118 ). ( B ) Expression of GFP-tagged Drosophila STING (under the native dSTING promoter) in 5 days old adult organs and tissues of dSTINGΔ;GFP-dSTING flies. Ten micrograms of extract was loaded per well. GFP-dSTING was detected using anti-GFP antibody. ( C ) modENCODE tissue expression data for adult tissues ( Brown et al., 2014 ). ( D ) Expression of GFP-tagged Drosophila STING (under the native dSTING promoter) in dSTINGΔ;GFP-dSTING third-instar larvae organs and tissues. Ten micrograms of extract was loaded per well. GFP-dSTING was detected using anti-GFP antibody. In gut samples, GFP-dSTING was detected as a double band, probably reflecting the proteolysis by gut enzymes. ( E ) modENCODE tissue expression data for larval tissues ( Brown et al., 2014 ). Source file for GFP-dSTING expression. Source file for GFP-dSTING expression (adult tissues). Source file for GFP-dSTING expression (larval tissues).
    Figure Legend Snippet: GFP-tagged Drosophila STING expression in different tissues. ( A ) Transcription of dSTING in wild type ( w 1118 ), mutant ( dSTINGΔ ), or ‘rescued’ ( dSTINGΔ;GFP-dSTING ) flies was measured by RT-qPCR. The bars indicate dSTING relative expression levels. Values were normalized to the wild type ( w 1118 ). ( B ) Expression of GFP-tagged Drosophila STING (under the native dSTING promoter) in 5 days old adult organs and tissues of dSTINGΔ;GFP-dSTING flies. Ten micrograms of extract was loaded per well. GFP-dSTING was detected using anti-GFP antibody. ( C ) modENCODE tissue expression data for adult tissues ( Brown et al., 2014 ). ( D ) Expression of GFP-tagged Drosophila STING (under the native dSTING promoter) in dSTINGΔ;GFP-dSTING third-instar larvae organs and tissues. Ten micrograms of extract was loaded per well. GFP-dSTING was detected using anti-GFP antibody. In gut samples, GFP-dSTING was detected as a double band, probably reflecting the proteolysis by gut enzymes. ( E ) modENCODE tissue expression data for larval tissues ( Brown et al., 2014 ). Source file for GFP-dSTING expression. Source file for GFP-dSTING expression (adult tissues). Source file for GFP-dSTING expression (larval tissues).

    Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR

    9) Product Images from "A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells"

    Article Title: A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells

    Journal: eLife

    doi: 10.7554/eLife.64444

    β-Catenin binding sites near ( A ) Pla2g7 and ( B ) Tafa5, two β-catenin target genes reported in Karner et al., 2011 . The ChIP-qPCR target sites were marked as red bins on the top track. The highlighted region is where our data is consistent with Karner’s.
    Figure Legend Snippet: β-Catenin binding sites near ( A ) Pla2g7 and ( B ) Tafa5, two β-catenin target genes reported in Karner et al., 2011 . The ChIP-qPCR target sites were marked as red bins on the top track. The highlighted region is where our data is consistent with Karner’s.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Supplementary RNA-Seq data analysis. ( A ) Overview of experiment design and data available (gray). ( B ) Bar plots show RT-qPCR measurement of relative expression of the indicated genes, as verification of results in Figure 1C . ( C ) Hierarchical cluster of R-square values between transcriptome-wide TPM of the indicated pair of replicate RNA-Seq data sets. ( D ) Top five enriched Gene Ontology (GO) terms of genes differentially expressed between low CHIR99021 (CHIR) condition and uncultured nephron progenitor cell (NPC). ( E ) Top five enriched GO terms of genes differentially expressed between low CHIR and no CHIR conditions. ( F ) Venn diagram shows overlap of β-catenin target genes with all genes associated to the GO term ‘cell cycle’ that are highly expressed in low CHIR versus no CHIR condition. ( G ) Heatmap and hierarchical cluster showing log2 relative TPM (TPM divided by mean across samples) to reflect change of gene expression (at isoform level) of Karner et al., 2011 ) class II genes (differentially expressed low CHIR > no CHIR or high CHIR > low CHIR) in our data set. The highlighted gene is supported by genetic evidence of regulation by β-catenin in the current data. ( H ) Immunofluorescence (IF) staining of SIX2 in unpurified NPC-free cortex (NFC) cells and NPC freshly purified from NFC; bar plots showing percentage of SIX2+ cells in the preps.
    Figure Legend Snippet: Supplementary RNA-Seq data analysis. ( A ) Overview of experiment design and data available (gray). ( B ) Bar plots show RT-qPCR measurement of relative expression of the indicated genes, as verification of results in Figure 1C . ( C ) Hierarchical cluster of R-square values between transcriptome-wide TPM of the indicated pair of replicate RNA-Seq data sets. ( D ) Top five enriched Gene Ontology (GO) terms of genes differentially expressed between low CHIR99021 (CHIR) condition and uncultured nephron progenitor cell (NPC). ( E ) Top five enriched GO terms of genes differentially expressed between low CHIR and no CHIR conditions. ( F ) Venn diagram shows overlap of β-catenin target genes with all genes associated to the GO term ‘cell cycle’ that are highly expressed in low CHIR versus no CHIR condition. ( G ) Heatmap and hierarchical cluster showing log2 relative TPM (TPM divided by mean across samples) to reflect change of gene expression (at isoform level) of Karner et al., 2011 ) class II genes (differentially expressed low CHIR > no CHIR or high CHIR > low CHIR) in our data set. The highlighted gene is supported by genetic evidence of regulation by β-catenin in the current data. ( H ) Immunofluorescence (IF) staining of SIX2 in unpurified NPC-free cortex (NFC) cells and NPC freshly purified from NFC; bar plots showing percentage of SIX2+ cells in the preps.

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Purification

    Supplementary evidence for differential expression of TCF/LEF factors. ( A ) Bar plots show RT-qPCR measurement of relative expression of TCF/LEF family factors, as verification for results in Figure 3A . ( B ) Bar plots show expression of individual transcripts of TCF/LEF factors in our RNA-Seq data. ( C ) tSNE plot displaying unbiased cluster of nephron lineage cells profiled by single-cell RNA-Seq. ( D ) Feature plots displaying distribution of self-renewal (red) and differentiation (green) marker genes transcripts on the tSNE plot. ( E ) Feature plots showing distribution of TCF/LEF factors transcripts on the tSNE plot. ( F ) Dot plots showing accumulated expression level of marker genes as well as TCF/LEF factor in selected clusters of cells. NPC: nephron progenitor cell; NFC: NPC-free cortex; CHIR: CHIR99021.
    Figure Legend Snippet: Supplementary evidence for differential expression of TCF/LEF factors. ( A ) Bar plots show RT-qPCR measurement of relative expression of TCF/LEF family factors, as verification for results in Figure 3A . ( B ) Bar plots show expression of individual transcripts of TCF/LEF factors in our RNA-Seq data. ( C ) tSNE plot displaying unbiased cluster of nephron lineage cells profiled by single-cell RNA-Seq. ( D ) Feature plots displaying distribution of self-renewal (red) and differentiation (green) marker genes transcripts on the tSNE plot. ( E ) Feature plots showing distribution of TCF/LEF factors transcripts on the tSNE plot. ( F ) Dot plots showing accumulated expression level of marker genes as well as TCF/LEF factor in selected clusters of cells. NPC: nephron progenitor cell; NFC: NPC-free cortex; CHIR: CHIR99021.

    Techniques Used: Expressing, Quantitative RT-PCR, RNA Sequencing Assay, Marker

    10) Product Images from "Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR"

    Article Title: Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR

    Journal: eLife

    doi: 10.7554/eLife.66834

    Cell concentration of 400 cells/μl, DNase treatment, and multiplexing PCR result in accurate OIL-PCR results. ( a ) Diagram of the Taqman assay used to monitor OIL-PCR results. Briefly, Taqman probes were designed to be complementary for the 16S rRNA genes in either E. coli or V. cholerae , each with its own fluorophore. OIL-PCR was performed on E. coli carrying the cmR gene on the pBAD33 plasmid but not present in V. cholerae. Fusion PCR products were recovered and nested probe-based qPCR was performed. Upon amplification of the gene, the probe is cleaved by Taq polymerase releasing the fluorophore from the quencher. Specific amplification of the designated region is measured by fluorescence of the expected fusion product vs the non-specific product. ( b ) OIL-PCR with primers targeting a plasmid-borne cmR gene was performed with a 1:1 mix of cmR positive E. coli and cmR negative V. cholerae cell suspensions with. A gradient of cell concentrations was tested (400–40,000 cells/μl), in addition to E. coli and V. cholerae suspensions alone as positive and negative controls. Control emulsions were mixed 1:1 after emulsification to test for droplet coalescence. ( c ) OIL-PCR with primers targeting a plasmid-borne cmR was performed after pretreating cells with (right) and without (left) dsDNase at two different 1:1 E. coli to V. cholerae cell suspension concentrations as well as on the individual bacterial strains for controls. ( d ) Multiplexed OIL-PCR was performed with primer sets targeting a genomic bla CTX-M gene in E. coli and a plasmid-borne bla TEM gene in V. cholerae. Experiments were performed in triplicate and on each of the organisms separately. Results are shown for the bla CTX-M (left) and bla TEM (right).
    Figure Legend Snippet: Cell concentration of 400 cells/μl, DNase treatment, and multiplexing PCR result in accurate OIL-PCR results. ( a ) Diagram of the Taqman assay used to monitor OIL-PCR results. Briefly, Taqman probes were designed to be complementary for the 16S rRNA genes in either E. coli or V. cholerae , each with its own fluorophore. OIL-PCR was performed on E. coli carrying the cmR gene on the pBAD33 plasmid but not present in V. cholerae. Fusion PCR products were recovered and nested probe-based qPCR was performed. Upon amplification of the gene, the probe is cleaved by Taq polymerase releasing the fluorophore from the quencher. Specific amplification of the designated region is measured by fluorescence of the expected fusion product vs the non-specific product. ( b ) OIL-PCR with primers targeting a plasmid-borne cmR gene was performed with a 1:1 mix of cmR positive E. coli and cmR negative V. cholerae cell suspensions with. A gradient of cell concentrations was tested (400–40,000 cells/μl), in addition to E. coli and V. cholerae suspensions alone as positive and negative controls. Control emulsions were mixed 1:1 after emulsification to test for droplet coalescence. ( c ) OIL-PCR with primers targeting a plasmid-borne cmR was performed after pretreating cells with (right) and without (left) dsDNase at two different 1:1 E. coli to V. cholerae cell suspension concentrations as well as on the individual bacterial strains for controls. ( d ) Multiplexed OIL-PCR was performed with primer sets targeting a genomic bla CTX-M gene in E. coli and a plasmid-borne bla TEM gene in V. cholerae. Experiments were performed in triplicate and on each of the organisms separately. Results are shown for the bla CTX-M (left) and bla TEM (right).

    Techniques Used: Concentration Assay, Multiplexing, Polymerase Chain Reaction, TaqMan Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction, Amplification, Fluorescence, Transmission Electron Microscopy

    BSA and excess MgCl 2 improve the efficiency of OIL-PCR and Ready Lyse Lysozyme remains active in OIL-PCR master mix. ( a ) Sybr-based qPCR was performed on the cmR gene carried on pBAD33 with varying concentrations of lysozyme in the presence (orange) or absence (blue) of BSA. Higher 2 (50-Ct) values represent greater amplification. ( b ) Lysozyme activity against B. subtilis suspended in the OIL-PCR optimized reaction mix with (solid line) and without (dashed line) lysozyme. ( c ) Sybr-based qPCR was performed on the cmR gene carried on the pBAD33 plasmid in E. coli MG1655 cells at increasing cell concentrations with (orange) and without (blue) additional MgCl 2 . Higher 2 (50-Ct) values represent greater amplification.
    Figure Legend Snippet: BSA and excess MgCl 2 improve the efficiency of OIL-PCR and Ready Lyse Lysozyme remains active in OIL-PCR master mix. ( a ) Sybr-based qPCR was performed on the cmR gene carried on pBAD33 with varying concentrations of lysozyme in the presence (orange) or absence (blue) of BSA. Higher 2 (50-Ct) values represent greater amplification. ( b ) Lysozyme activity against B. subtilis suspended in the OIL-PCR optimized reaction mix with (solid line) and without (dashed line) lysozyme. ( c ) Sybr-based qPCR was performed on the cmR gene carried on the pBAD33 plasmid in E. coli MG1655 cells at increasing cell concentrations with (orange) and without (blue) additional MgCl 2 . Higher 2 (50-Ct) values represent greater amplification.

    Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Amplification, Activity Assay, Plasmid Preparation

    11) Product Images from "Toll-like receptor 4 signaling activates ERG function in prostate cancer and provides a therapeutic target"

    Article Title: Toll-like receptor 4 signaling activates ERG function in prostate cancer and provides a therapeutic target

    Journal: Nar Cancer

    doi: 10.1093/narcan/zcaa046

    TLR4 is an important component of ERG-mediated migration. ( A ) ERG-positive (RWPE-ERG) and ERG-negative (RWPE-KRAS) prostate cells were subjected to a migration-based shRNA screen using a library from Addgene/Cellecta. ( B ) Trans -well migration of non-migratory and migratory cells. Cells removed from within the insert after migration were classified as non-migratory cells, and cells removed from the underside of the insert were classified as migratory. These cells were then tested in a second round of migration. ( C ) Enrichr pathway analysis output for the top 5% of over-represented genes in non-migratory ERG-positive cells. ( D ) Correlation of ERG and TLR4 mRNA expression in prostate cancer patient samples visualized via cBioPortal. ( E ) Expression of TLR4 mRNA in normal prostate versus prostate cancer samples. ( F ) Relative mRNA levels by RNA-seq of TLR4 and three endogenous ligands in RWPE-ERG normalized to RWPE-empty vector. ( G ) RWPE-ERG mRNA level of TLR4 and endogenous ligands by RT-qPCR normalized to RWPE-empty vector cells. ( H ) TLR4 protein expression across a panel of ERG-positive and ERG-negative prostate cell lines. ( I ) Secreted protein expression of BGN and Hsc70 (gene name HSPA8) in RWPE-empty vector and RWPE-ERG conditioned media. Tubulin expression from the corresponding whole cell extracts is shown as a loading control. Shown are mean and SEM of three biological replicates and ** indicates P
    Figure Legend Snippet: TLR4 is an important component of ERG-mediated migration. ( A ) ERG-positive (RWPE-ERG) and ERG-negative (RWPE-KRAS) prostate cells were subjected to a migration-based shRNA screen using a library from Addgene/Cellecta. ( B ) Trans -well migration of non-migratory and migratory cells. Cells removed from within the insert after migration were classified as non-migratory cells, and cells removed from the underside of the insert were classified as migratory. These cells were then tested in a second round of migration. ( C ) Enrichr pathway analysis output for the top 5% of over-represented genes in non-migratory ERG-positive cells. ( D ) Correlation of ERG and TLR4 mRNA expression in prostate cancer patient samples visualized via cBioPortal. ( E ) Expression of TLR4 mRNA in normal prostate versus prostate cancer samples. ( F ) Relative mRNA levels by RNA-seq of TLR4 and three endogenous ligands in RWPE-ERG normalized to RWPE-empty vector. ( G ) RWPE-ERG mRNA level of TLR4 and endogenous ligands by RT-qPCR normalized to RWPE-empty vector cells. ( H ) TLR4 protein expression across a panel of ERG-positive and ERG-negative prostate cell lines. ( I ) Secreted protein expression of BGN and Hsc70 (gene name HSPA8) in RWPE-empty vector and RWPE-ERG conditioned media. Tubulin expression from the corresponding whole cell extracts is shown as a loading control. Shown are mean and SEM of three biological replicates and ** indicates P

    Techniques Used: Migration, shRNA, Expressing, RNA Sequencing Assay, Plasmid Preparation, Quantitative RT-PCR

    12) Product Images from "Utility of fungal polymerase chain reaction on nasal swab samples in the diagnosis and monitoring of sinonasal aspergillosis in dogs. Utility of fungal polymerase chain reaction on nasal swab samples in the diagnosis and monitoring of sinonasal aspergillosis in dogs"

    Article Title: Utility of fungal polymerase chain reaction on nasal swab samples in the diagnosis and monitoring of sinonasal aspergillosis in dogs. Utility of fungal polymerase chain reaction on nasal swab samples in the diagnosis and monitoring of sinonasal aspergillosis in dogs

    Journal: Journal of Veterinary Internal Medicine

    doi: 10.1111/jvim.16441

    scattergram of qPCR Aspfum ( Aspergillus fumigatus ) test results, expressed as Ct value, in the 4 groups of dogs: SNA (sinonasal aspergillosis), cured SNA, Non‐SNA nasal disease and healthy. Dogs with SNA had a mean Ct significantly lower than in the 3 other groups ( P
    Figure Legend Snippet: scattergram of qPCR Aspfum ( Aspergillus fumigatus ) test results, expressed as Ct value, in the 4 groups of dogs: SNA (sinonasal aspergillosis), cured SNA, Non‐SNA nasal disease and healthy. Dogs with SNA had a mean Ct significantly lower than in the 3 other groups ( P

    Techniques Used: Real-time Polymerase Chain Reaction

    13) Product Images from "Sulforaphane diminishes moonlighting of pyruvate kinase M2 and interleukin 1β expression in M1 (LPS) macrophages"

    Article Title: Sulforaphane diminishes moonlighting of pyruvate kinase M2 and interleukin 1β expression in M1 (LPS) macrophages

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2022.935692

    Sfn does not markedly interfere with M2 marker expression during macrophage polarization initiated by IL-4. IBMDM macrophages were pretreated with DMSO or with the indicated concentrations of Sfn for 30 min before they were stimulated with IL-4 (20 ng/ml) for 24 h. RNA was extracted, reversely transcribed, and subjected to qPCR analysis for mrc1 (A) , mgl1 (B) , mgl2 (C) , arg1 (D) , and cd36 (E) mRNA expression. Ppia served as a reference gene; data were referred to as M0 controls. Production of total polyamines (F) was assessed by a commercial kit and TGF-β (G) by an ELISA as described in the Methods section. Data depict mean ± SD from at least three independent biological replicates ( * p
    Figure Legend Snippet: Sfn does not markedly interfere with M2 marker expression during macrophage polarization initiated by IL-4. IBMDM macrophages were pretreated with DMSO or with the indicated concentrations of Sfn for 30 min before they were stimulated with IL-4 (20 ng/ml) for 24 h. RNA was extracted, reversely transcribed, and subjected to qPCR analysis for mrc1 (A) , mgl1 (B) , mgl2 (C) , arg1 (D) , and cd36 (E) mRNA expression. Ppia served as a reference gene; data were referred to as M0 controls. Production of total polyamines (F) was assessed by a commercial kit and TGF-β (G) by an ELISA as described in the Methods section. Data depict mean ± SD from at least three independent biological replicates ( * p

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

    DOG or the LDH inhibitor FX11 cannot attenuate inhibition of IL-1β expression by Sfn. iBMDM were left untreated (M0), treated with LPS (25 ng/ml) (M1) and (A) LPS + DOG (2 mM) or (B) LPS + FX11 (10 µM) for 6 h before relative mRNA expression of IL-1β was assessed by qPCR (using ppia as reference gene). iBMDM were pretreated with DMSO or Sfn (1 or 3 µM) for 30 min in the absence or presence of either (C) 2 mM (DOG) or (D) 10 µM FX11 before LPS (25 ng/ml) was added for 6 h. Then mRNA expression of il1β was assessed by qPCR (using ppia as reference gene) and referred to the respective DMSO controls. (E) Murine macrophages were pretreated with DMSO or Sfn (3 µM) for 30 min in the absence or presence of 2 mM DOG before LPS was added for another 6 h. After protein extraction total cell lysates were then subjected to immunoblot analysis for pro-IL-1β and actin as a loading control. Representative blots are shown and bar graphs depict compiled densitometric data from three biological replicates (mean ± SD, n = 3; * p
    Figure Legend Snippet: DOG or the LDH inhibitor FX11 cannot attenuate inhibition of IL-1β expression by Sfn. iBMDM were left untreated (M0), treated with LPS (25 ng/ml) (M1) and (A) LPS + DOG (2 mM) or (B) LPS + FX11 (10 µM) for 6 h before relative mRNA expression of IL-1β was assessed by qPCR (using ppia as reference gene). iBMDM were pretreated with DMSO or Sfn (1 or 3 µM) for 30 min in the absence or presence of either (C) 2 mM (DOG) or (D) 10 µM FX11 before LPS (25 ng/ml) was added for 6 h. Then mRNA expression of il1β was assessed by qPCR (using ppia as reference gene) and referred to the respective DMSO controls. (E) Murine macrophages were pretreated with DMSO or Sfn (3 µM) for 30 min in the absence or presence of 2 mM DOG before LPS was added for another 6 h. After protein extraction total cell lysates were then subjected to immunoblot analysis for pro-IL-1β and actin as a loading control. Representative blots are shown and bar graphs depict compiled densitometric data from three biological replicates (mean ± SD, n = 3; * p

    Techniques Used: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Protein Extraction

    Sfn impedes M1 marker expression during macrophage polarization initiated by LPS. IBMDM macrophages were pretreated with DMSO or the indicated concentrations of Sfn for 30 min before they were stimulated with LPS (25 ng/ml) for 24 h. RNA was extracted, reversely transcribed, and subjected to qPCR analysis for il1β (A) , il6 (B) , and nos2 (C) mRNA expression. Ppia served as a reference gene; data were referred to as M0 controls. Secretion of TNF-α (D) was assessed using an ELISA, and nitric oxide (E) was measured by monitoring nitrite levels via the Griess assay as described in the Methods section. Levels of intracellular ROS (F) were determined by H 2 DCF-DA staining and subsequent flow cytometric analysis. Data depict mean ± SD from at least three independent biological replicates ( * p
    Figure Legend Snippet: Sfn impedes M1 marker expression during macrophage polarization initiated by LPS. IBMDM macrophages were pretreated with DMSO or the indicated concentrations of Sfn for 30 min before they were stimulated with LPS (25 ng/ml) for 24 h. RNA was extracted, reversely transcribed, and subjected to qPCR analysis for il1β (A) , il6 (B) , and nos2 (C) mRNA expression. Ppia served as a reference gene; data were referred to as M0 controls. Secretion of TNF-α (D) was assessed using an ELISA, and nitric oxide (E) was measured by monitoring nitrite levels via the Griess assay as described in the Methods section. Levels of intracellular ROS (F) were determined by H 2 DCF-DA staining and subsequent flow cytometric analysis. Data depict mean ± SD from at least three independent biological replicates ( * p

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

    14) Product Images from "Kinship of conditionally immortalized cells derived from fetal bone to human bone-derived mesenchymal stroma cells"

    Article Title: Kinship of conditionally immortalized cells derived from fetal bone to human bone-derived mesenchymal stroma cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-90161-2

    Differentiation of hFOB spheroids. ( A ) Spheroids were obtained using low attachment plates. 1000 cells were seeded per well and allowed to aggregate at the bottom of the plates for 48 h at 34 °C. Thereafter, plates were incubated for 3 days at 34 °C (upper panel) or at 39 °C (lower panel). Spheroids were stained with Calcein-AM (Calcein) and calcium deposition was visualized with Xylenol Orange (XO). For adipogenic differentiation ( C ) spheroids were incubated in adipogenic medium at 34 °C (upper panel) or 39 °C (lower panel) for 3 days. Cells were live-stained with Calcein and Autodot (pink). Images were taken using a DMi8 microscope (Leica) and acquisition analysis was performed with LAS X Software. (B, D) Quantitative PCR for the genes osteocalcin and PPARγ was performed to confirm osteogenic or adipogenic differentiation at day 4 post induction. Relative expression was normalized to hFOB cells grown in 2D at 34 °C.
    Figure Legend Snippet: Differentiation of hFOB spheroids. ( A ) Spheroids were obtained using low attachment plates. 1000 cells were seeded per well and allowed to aggregate at the bottom of the plates for 48 h at 34 °C. Thereafter, plates were incubated for 3 days at 34 °C (upper panel) or at 39 °C (lower panel). Spheroids were stained with Calcein-AM (Calcein) and calcium deposition was visualized with Xylenol Orange (XO). For adipogenic differentiation ( C ) spheroids were incubated in adipogenic medium at 34 °C (upper panel) or 39 °C (lower panel) for 3 days. Cells were live-stained with Calcein and Autodot (pink). Images were taken using a DMi8 microscope (Leica) and acquisition analysis was performed with LAS X Software. (B, D) Quantitative PCR for the genes osteocalcin and PPARγ was performed to confirm osteogenic or adipogenic differentiation at day 4 post induction. Relative expression was normalized to hFOB cells grown in 2D at 34 °C.

    Techniques Used: Incubation, Staining, Microscopy, Software, Real-time Polymerase Chain Reaction, Expressing

    15) Product Images from "EP400NL is required for cMyc-mediated PD-L1 gene activation by forming a transcriptional coactivator complex"

    Article Title: EP400NL is required for cMyc-mediated PD-L1 gene activation by forming a transcriptional coactivator complex

    Journal: bioRxiv

    doi: 10.1101/2021.05.30.446361

    EP400NL is recruited at the PD-L1 promoter in a cMyc dependent manner (A) Enrichment of cMyc, EP400NL, BRG1, and RuvBL2 at the PD-L1 promoter of Flp-In™ T-REx™ cell line stably expressing tetracycline-inducible EP400NL. The purified DNA after ChIP reactions was analyzed by qPCR over the regions of PD-L1 promoter (Myc binding site) or GAPDH promoter. Four combinations of the experimental conditions (the presence and absence of serum and tetracycline: ±Ser/±Tet) were used in the ChIP analyses [Two-way ANOVA, F (1,8) = 36.01, p = 0.0003 (cMyc), F (1,8) = 263.7, p
    Figure Legend Snippet: EP400NL is recruited at the PD-L1 promoter in a cMyc dependent manner (A) Enrichment of cMyc, EP400NL, BRG1, and RuvBL2 at the PD-L1 promoter of Flp-In™ T-REx™ cell line stably expressing tetracycline-inducible EP400NL. The purified DNA after ChIP reactions was analyzed by qPCR over the regions of PD-L1 promoter (Myc binding site) or GAPDH promoter. Four combinations of the experimental conditions (the presence and absence of serum and tetracycline: ±Ser/±Tet) were used in the ChIP analyses [Two-way ANOVA, F (1,8) = 36.01, p = 0.0003 (cMyc), F (1,8) = 263.7, p

    Techniques Used: Stable Transfection, Expressing, Purification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    16) Product Images from "OxPhos Dysfunction Causes Hypermetabolism and Reduces Lifespan in Cells and in Patients with Mitochondrial Diseases"

    Article Title: OxPhos Dysfunction Causes Hypermetabolism and Reduces Lifespan in Cells and in Patients with Mitochondrial Diseases

    Journal: bioRxiv

    doi: 10.1101/2021.11.29.470428

    OxPhos defects trigger hypersecretion of metabokines and age-related cytokines. ( A ) Cytokine dynamics across the lifespan measured on two multiplex (Luminex) arrays. Cytokine levels are normalized to the number of cells at the time of sampling, shown as Log 2 median-centered for each cytokine; samples with undetectable values are shown as grey cells. Columns represent repeated-measures (n=6-8) along the lifespan of each controls and SURF1 donor (n=3 per group). ( B ) Comparison of maximum cytokine concentration reached in each of the SURF1 and healthy control donors, showing general upregulation of most metabokines and cytokines. The value for TGF-⍺ is heavily influenced by a single very high value in Donor 3. ( C ) Cell-free GDF15 time course as measured on the Cytokine array. Inset compares early release between 20-80 days. ( D ) Media GDF15 levels across the cellular lifespan measured by enzyme-linked immunosorbent assay (ELISA), normalized to the number of cells at time of sampling. Samples with non-detectable values (N.D.) are shown as zero values. (E) Media IL-6 levels across the cellular lifespan by ELISA, normalized to the number of cells at time of sampling. (F) Cell-free mitochondrial DNA dynamics across the cellular lifespan using qPCR, normalized to the number of cells at time of sampling. n = 3 per group, 6-13 timepoints per condition. Data are means ± SEM. * P
    Figure Legend Snippet: OxPhos defects trigger hypersecretion of metabokines and age-related cytokines. ( A ) Cytokine dynamics across the lifespan measured on two multiplex (Luminex) arrays. Cytokine levels are normalized to the number of cells at the time of sampling, shown as Log 2 median-centered for each cytokine; samples with undetectable values are shown as grey cells. Columns represent repeated-measures (n=6-8) along the lifespan of each controls and SURF1 donor (n=3 per group). ( B ) Comparison of maximum cytokine concentration reached in each of the SURF1 and healthy control donors, showing general upregulation of most metabokines and cytokines. The value for TGF-⍺ is heavily influenced by a single very high value in Donor 3. ( C ) Cell-free GDF15 time course as measured on the Cytokine array. Inset compares early release between 20-80 days. ( D ) Media GDF15 levels across the cellular lifespan measured by enzyme-linked immunosorbent assay (ELISA), normalized to the number of cells at time of sampling. Samples with non-detectable values (N.D.) are shown as zero values. (E) Media IL-6 levels across the cellular lifespan by ELISA, normalized to the number of cells at time of sampling. (F) Cell-free mitochondrial DNA dynamics across the cellular lifespan using qPCR, normalized to the number of cells at time of sampling. n = 3 per group, 6-13 timepoints per condition. Data are means ± SEM. * P

    Techniques Used: Multiplex Assay, Luminex, Sampling, Concentration Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    Meta-analysis of human studies reveals increased energy expenditure and shortened lifespan in primary mitochondrial diseases. ( A ) Overall conceptual model linking mtDNA- and nDNA-related OxPhos defects to impaired metabolic efficiency at the cellular level, impacting whole-body resting energy expenditure and clinical outcomes. ( B ) Skeletal muscle biopsy with individual muscle fibers stained with cytochrome c oxidase/succinate dehydrogenase (COX/SDH) histochemistry to reveal functional (brown) and respiratory chain deficient (blue) mitochondria. In the affected cell (middle), three sub-regions showing low, intermediate, and high mtDNA mutation load were captured by laser capture microdissection and subjected to quantitative PCR analysis as in (Picard et al. 2012). Subcellular regions with high mtDNA mutation load show elevated mtDNA density, which is predicted to increase the energetic cost due to maintenance and turnover processes. WT , wild type. ( C ) Meta- analysis of human mitochondrial disease cohorts showing elevated resting heart rate (n=104 controls, 111 patients), ( D ) catecholamines (urinary-Cohort 3 and blood-Cohort 6) at rest or during fixed-intensity exercise (n=38 controls, 19 patients), ( E ) whole-body oxygen consumption measured by indirect calorimetry at rest or during response to mild exercise challenge; 1 before training, 2 after training. Slope refers to the rate of increase in VO 2 relative to work rate, where a higher slope indicates increased energetic cost for a given work rate (n=56 controls, 78 patients). ( F ) Body mass index (BMI) across mitochondrial disease cohorts and compared to national averages (USA, UK, Italy) (n=285 controls, 174 patients). ( G ) Average life expectancy in individuals with mitochondrial diseases relative to national averages (n=301 patients). Data are means ± SEM, with % difference between mitochondrial disease and control group where available. ( H ) Mortality (age of death) over 10 years (2010-2020) in Cohort 17 compared to national averages for women and men (n=109 patients). See Table 1 for cohort details. Total n=225 healthy controls, 690 patients. Groups compared by paired t tests (C and F) or one-sample t tests (D and E), * p
    Figure Legend Snippet: Meta-analysis of human studies reveals increased energy expenditure and shortened lifespan in primary mitochondrial diseases. ( A ) Overall conceptual model linking mtDNA- and nDNA-related OxPhos defects to impaired metabolic efficiency at the cellular level, impacting whole-body resting energy expenditure and clinical outcomes. ( B ) Skeletal muscle biopsy with individual muscle fibers stained with cytochrome c oxidase/succinate dehydrogenase (COX/SDH) histochemistry to reveal functional (brown) and respiratory chain deficient (blue) mitochondria. In the affected cell (middle), three sub-regions showing low, intermediate, and high mtDNA mutation load were captured by laser capture microdissection and subjected to quantitative PCR analysis as in (Picard et al. 2012). Subcellular regions with high mtDNA mutation load show elevated mtDNA density, which is predicted to increase the energetic cost due to maintenance and turnover processes. WT , wild type. ( C ) Meta- analysis of human mitochondrial disease cohorts showing elevated resting heart rate (n=104 controls, 111 patients), ( D ) catecholamines (urinary-Cohort 3 and blood-Cohort 6) at rest or during fixed-intensity exercise (n=38 controls, 19 patients), ( E ) whole-body oxygen consumption measured by indirect calorimetry at rest or during response to mild exercise challenge; 1 before training, 2 after training. Slope refers to the rate of increase in VO 2 relative to work rate, where a higher slope indicates increased energetic cost for a given work rate (n=56 controls, 78 patients). ( F ) Body mass index (BMI) across mitochondrial disease cohorts and compared to national averages (USA, UK, Italy) (n=285 controls, 174 patients). ( G ) Average life expectancy in individuals with mitochondrial diseases relative to national averages (n=301 patients). Data are means ± SEM, with % difference between mitochondrial disease and control group where available. ( H ) Mortality (age of death) over 10 years (2010-2020) in Cohort 17 compared to national averages for women and men (n=109 patients). See Table 1 for cohort details. Total n=225 healthy controls, 690 patients. Groups compared by paired t tests (C and F) or one-sample t tests (D and E), * p

    Techniques Used: Staining, Functional Assay, Mutagenesis, Laser Capture Microdissection, Real-time Polymerase Chain Reaction

    17) Product Images from "β1 integrin, ILK and mTOR regulate collagen synthesis in mechanically loaded tendon cells"

    Article Title: β1 integrin, ILK and mTOR regulate collagen synthesis in mechanically loaded tendon cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-69267-6

    Role of mTOR pathway in regulation of collagen expression. ( a ) Immunoblot analysis of tendon-derived stromal cells after incubation with AKT inhibitor (GSK2141795) and mTOR inhibitors (INK128, PP247 and Torin) for 48 h. ( b ) In-cell western analysis of human tendon cells after 48 h and 72 h incubation with GSK2141795 and mTOR inhibitors. ( c ) Densitometry analysis of the in-cell western in b . ( d–g ) qRT-PCR analysis of gene expression in human tendon cells after 48 h of incubation with GSK2141795 ( d ), INK128 ( e ), Torin ( f ), and PP247 ( g ). Increasing ΔCt indicates decreasing gene expression. Two-way and one-way ANOVA followed by Bonferroni's multiple comparisons test for data of in-cell western densitometry and qPCR, respectively.; mean ± SE; ns P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; n ≥ 3 biological replicates.
    Figure Legend Snippet: Role of mTOR pathway in regulation of collagen expression. ( a ) Immunoblot analysis of tendon-derived stromal cells after incubation with AKT inhibitor (GSK2141795) and mTOR inhibitors (INK128, PP247 and Torin) for 48 h. ( b ) In-cell western analysis of human tendon cells after 48 h and 72 h incubation with GSK2141795 and mTOR inhibitors. ( c ) Densitometry analysis of the in-cell western in b . ( d–g ) qRT-PCR analysis of gene expression in human tendon cells after 48 h of incubation with GSK2141795 ( d ), INK128 ( e ), Torin ( f ), and PP247 ( g ). Increasing ΔCt indicates decreasing gene expression. Two-way and one-way ANOVA followed by Bonferroni's multiple comparisons test for data of in-cell western densitometry and qPCR, respectively.; mean ± SE; ns P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; n ≥ 3 biological replicates.

    Techniques Used: Expressing, Derivative Assay, Incubation, In-Cell ELISA, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    18) Product Images from "The trypanosome Variant Surface Glycoprotein mRNA is stabilized by an essential unconventional RNA-binding protein"

    Article Title: The trypanosome Variant Surface Glycoprotein mRNA is stabilized by an essential unconventional RNA-binding protein

    Journal: bioRxiv

    doi: 10.1101/2020.10.08.331769

    RNA antisense purification identifies proteins that interact directly with VSG mRNA A. A schematic overview of the method. Bloodstream-form T brucei were subjected to UV irradiation. After cell lysis, the lysate was incubated with streptavidin-coated magnetic beads. The unbound portion was then incubated with biotinylated 90 nt long DNA probes, to hybridize to either alpha-tubulin (TUB) or VSG2 mRNA, then probe-target complexes were captured by streptavidin-coated magnetic beads. To decrease background, samples were treated with DNase I. The supernatant was collected, and the second set of ribonucleoprotein complexes (VSG2 or TUB) was captured in a similar way. Protein and RNA were eluted from the beads, and subjected to mass spectrometry (LC-MS/MS) for protein identification and RT-qPCR for relative RNA quantification. B. Enrichment of VSG2 and TUB transcripts after RNA antisense purification. The individual data points show RNA levels relative to rRNA for each independent pulldown, measured by RT-qPCR, and bars represent means. Experiment 1 includes some preparations that were purified using only VSG. Before mass spectrometry of VSG or TUB preparations from each experiment, the material for all pull-downs shown was pooled. C. The triplicate experiments identified proteins that reproducibly enriched with VSG2 relative to TUB, and vice-versa. Proteins significantly enriched (FDR 1%; s0=0.1) are filled dark grey. Proteins associated with the MKT1 complex are in pink and CFB2 is cyan. The data for this graph are in Supplementary Table S2, sheet 2.
    Figure Legend Snippet: RNA antisense purification identifies proteins that interact directly with VSG mRNA A. A schematic overview of the method. Bloodstream-form T brucei were subjected to UV irradiation. After cell lysis, the lysate was incubated with streptavidin-coated magnetic beads. The unbound portion was then incubated with biotinylated 90 nt long DNA probes, to hybridize to either alpha-tubulin (TUB) or VSG2 mRNA, then probe-target complexes were captured by streptavidin-coated magnetic beads. To decrease background, samples were treated with DNase I. The supernatant was collected, and the second set of ribonucleoprotein complexes (VSG2 or TUB) was captured in a similar way. Protein and RNA were eluted from the beads, and subjected to mass spectrometry (LC-MS/MS) for protein identification and RT-qPCR for relative RNA quantification. B. Enrichment of VSG2 and TUB transcripts after RNA antisense purification. The individual data points show RNA levels relative to rRNA for each independent pulldown, measured by RT-qPCR, and bars represent means. Experiment 1 includes some preparations that were purified using only VSG. Before mass spectrometry of VSG or TUB preparations from each experiment, the material for all pull-downs shown was pooled. C. The triplicate experiments identified proteins that reproducibly enriched with VSG2 relative to TUB, and vice-versa. Proteins significantly enriched (FDR 1%; s0=0.1) are filled dark grey. Proteins associated with the MKT1 complex are in pink and CFB2 is cyan. The data for this graph are in Supplementary Table S2, sheet 2.

    Techniques Used: Purification, Irradiation, Lysis, Incubation, Magnetic Beads, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Quantitative RT-PCR

    19) Product Images from "cGAS/STING-DEPENDENT SENSING OF ENDOGENOUS RNA"

    Article Title: cGAS/STING-DEPENDENT SENSING OF ENDOGENOUS RNA

    Journal: bioRxiv

    doi: 10.1101/2022.05.16.492039

    MDA5 drives spontaneous IFN production in a cGAS/STING-dependent manner in Samhd1 Δ/Δ mice. Related to Figure 4 . (A) Samhd1 +/+ and Samhd1 Δ/Δ mice were treated i.p. with 10 mg/kg/day H-151 or vehicle for 14 days. Transcript levels of the indicated ISGs were determined in spleen. Fold change compared with the WT - vehicle group is shown, n=4 in each group (Two-way ANOVA followed by Tukey’s multiple comparison test). (B) Post-replicative senescence Samhd1 Δ/Δ and Samhd1 +/+ MEFs were transduced with empty lentivirus or a lentivirus which expresses the cDNA of murine Samhd1 isoform1 as well as EYFP. Transduced cells were enriched by FACS for EYFP and transcript levels of the indicated ISGs were determined by qRT-PCR. Data of two independent measurements is displayed as fold change compared with the mean of Samhd1 +/+ MEFs transduced with empty lentivirus (Two-way ANOVA followed by Tukey’s multiple comparison test). (C) Relative transcript levels of the indicated ISGs measured by qRT-PCR in post-replicative senescence Samhd1 Δ/Δ MEFs with additional CRISPR-mediated inactivation of the genes cGas (n=4), Ifih1 (n=3) and Ddx58 (n=2) after lipofection with 1 μg/ml plasmid DNA (dsDNA), 100 ng/ml poly I:C, 100 ng/ml pppRNA or incubation with 10 μg/ml DMXAA for 16 hours. Fold change compared to Lipo-treated Samhd1 +/+ MEFs is shown. (D) Representative western blot for cGAS in GFP-cGas KI/KI and GFP-cGas WT/WT control mice (left). Data from two independent experiments for densitometric quantification of cGAS signal relative to the signal for β-actin (right, Student’s t test). cGAS = 62 kDa, GFP-cGAS around 92 kDa. (E) Spontaneous in vivo Ifnb1-luciferase signal in Samhd1 +/Δ (ctrl), Samhd1 Δ/Δ and Trexi KO/KO mice. All mice were homozygous for the luciferase knock in ( ΔβLUC KI/KI ). (F) Normalized read counts of ISG transcripts in Samhd1 Δ/Δ Ifih1 -/- vs. Samhd1 Δ/Δ Sting1 GT/GT mice. **=p
    Figure Legend Snippet: MDA5 drives spontaneous IFN production in a cGAS/STING-dependent manner in Samhd1 Δ/Δ mice. Related to Figure 4 . (A) Samhd1 +/+ and Samhd1 Δ/Δ mice were treated i.p. with 10 mg/kg/day H-151 or vehicle for 14 days. Transcript levels of the indicated ISGs were determined in spleen. Fold change compared with the WT - vehicle group is shown, n=4 in each group (Two-way ANOVA followed by Tukey’s multiple comparison test). (B) Post-replicative senescence Samhd1 Δ/Δ and Samhd1 +/+ MEFs were transduced with empty lentivirus or a lentivirus which expresses the cDNA of murine Samhd1 isoform1 as well as EYFP. Transduced cells were enriched by FACS for EYFP and transcript levels of the indicated ISGs were determined by qRT-PCR. Data of two independent measurements is displayed as fold change compared with the mean of Samhd1 +/+ MEFs transduced with empty lentivirus (Two-way ANOVA followed by Tukey’s multiple comparison test). (C) Relative transcript levels of the indicated ISGs measured by qRT-PCR in post-replicative senescence Samhd1 Δ/Δ MEFs with additional CRISPR-mediated inactivation of the genes cGas (n=4), Ifih1 (n=3) and Ddx58 (n=2) after lipofection with 1 μg/ml plasmid DNA (dsDNA), 100 ng/ml poly I:C, 100 ng/ml pppRNA or incubation with 10 μg/ml DMXAA for 16 hours. Fold change compared to Lipo-treated Samhd1 +/+ MEFs is shown. (D) Representative western blot for cGAS in GFP-cGas KI/KI and GFP-cGas WT/WT control mice (left). Data from two independent experiments for densitometric quantification of cGAS signal relative to the signal for β-actin (right, Student’s t test). cGAS = 62 kDa, GFP-cGAS around 92 kDa. (E) Spontaneous in vivo Ifnb1-luciferase signal in Samhd1 +/Δ (ctrl), Samhd1 Δ/Δ and Trexi KO/KO mice. All mice were homozygous for the luciferase knock in ( ΔβLUC KI/KI ). (F) Normalized read counts of ISG transcripts in Samhd1 Δ/Δ Ifih1 -/- vs. Samhd1 Δ/Δ Sting1 GT/GT mice. **=p

    Techniques Used: Mouse Assay, Transduction, FACS, Quantitative RT-PCR, CRISPR, Plasmid Preparation, Incubation, Western Blot, In Vivo, Luciferase, Knock-In

    MDA5 drives spontaneous IFN production in a cGAS/STING-dependent manner in Samhd1 Δ/Δ mice. For the whole figure - = homozygous null, + = homozygous wild type. (A) Enrichment of Reactome gene sets (MSigDB) in the transcriptome of peritoneal macrophages from mutant mice compared with littermate wild type controls of Samhd1 Δ/Δ mice. (B) Normalized read counts for the indicated ISG transcripts (left) and transcripts of the CELL CYCLE CHECKPOINTS gene set (right) from the analysis shown in (A). (C) Relative transcript levels of the indicated ISGs measured by qRT-PCR in post-replicative senescence Samhd1 Δ/Δ MEFs with additional CRISPR-mediated inactivation of the genes cGas (n=4), Ifih1 (n=3) and Ddx58 (n=2). Data of two independent experiments were pooled and displayed as fold change compared to the mean of Samhd1 +/+ MEFs (multiple t tests, summary of results is shown with p
    Figure Legend Snippet: MDA5 drives spontaneous IFN production in a cGAS/STING-dependent manner in Samhd1 Δ/Δ mice. For the whole figure - = homozygous null, + = homozygous wild type. (A) Enrichment of Reactome gene sets (MSigDB) in the transcriptome of peritoneal macrophages from mutant mice compared with littermate wild type controls of Samhd1 Δ/Δ mice. (B) Normalized read counts for the indicated ISG transcripts (left) and transcripts of the CELL CYCLE CHECKPOINTS gene set (right) from the analysis shown in (A). (C) Relative transcript levels of the indicated ISGs measured by qRT-PCR in post-replicative senescence Samhd1 Δ/Δ MEFs with additional CRISPR-mediated inactivation of the genes cGas (n=4), Ifih1 (n=3) and Ddx58 (n=2). Data of two independent experiments were pooled and displayed as fold change compared to the mean of Samhd1 +/+ MEFs (multiple t tests, summary of results is shown with p

    Techniques Used: Mouse Assay, Mutagenesis, Quantitative RT-PCR, CRISPR

    20) Product Images from "Analysis of Global Collection of Group A Streptococcus Genomes Reveals that the Majority Encode a Trio of M and M-Like Proteins"

    Article Title: Analysis of Global Collection of Group A Streptococcus Genomes Reveals that the Majority Encode a Trio of M and M-Like Proteins

    Journal: mSphere

    doi: 10.1128/mSphere.00806-19

    Expression analysis of Mga regulon genes. cDNA from 19 isolates grown to mid-log phase in rich medium were analyzed for the expression of Mga regulon genes. The isolates were selected to be representative of all possible Mga regulon configurations and emm cluster diversity where possible. Primers were designed to amplify all members of the gene family where possible ( mrp , enn , pgs , sph , and scpA ) and to amplify a subset where sequence diversity necessitates. The dot plot symbols represent the mean value of the four qPCR analyses for each isolate, and the error bars represent the standard errors for all isolates for each gene.
    Figure Legend Snippet: Expression analysis of Mga regulon genes. cDNA from 19 isolates grown to mid-log phase in rich medium were analyzed for the expression of Mga regulon genes. The isolates were selected to be representative of all possible Mga regulon configurations and emm cluster diversity where possible. Primers were designed to amplify all members of the gene family where possible ( mrp , enn , pgs , sph , and scpA ) and to amplify a subset where sequence diversity necessitates. The dot plot symbols represent the mean value of the four qPCR analyses for each isolate, and the error bars represent the standard errors for all isolates for each gene.

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

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    New England Biolabs luna universal qpcr master mix
    Generation of TUBL-deficient mice. (A) Schematic representation of the WT TINCR allele, the single-stranded oligodeoxynucleotide (ssODN), and the mutant allele after homologous recombination. Exons are denoted by numbered boxes. The single guide RNA (sgRNA) for the CRISPR-Cas9 system and its protospacer adjacent motif (PAM) are indicated by contiguous black and red underlines, respectively. The TUBL ORF is represented by the gray shading in the box corresponding to exon 1 of TINCR . (B) Predicted secondary structure and minimal free energy for WT TINCR and the mutant form generated by the CRISPR-Cas9 system for establishment of Tubl −/− mice. The triangle indicates the 5’ end of the transcript. (C) <t>PCR</t> analysis of genomic DNA from the tail of mice of the indicated genotypes. The PCR products were digested with EcoRI before electrophoresis. (D) <t>RT-qPCR</t> analysis of TINCR in the epidermis of Tubl +/+ and Tubl −/− mice. Data are means ± SD (n = 3 independent experiments). ***p
    Luna Universal Qpcr Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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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    Generation of TUBL-deficient mice. (A) Schematic representation of the WT TINCR allele, the single-stranded oligodeoxynucleotide (ssODN), and the mutant allele after homologous recombination. Exons are denoted by numbered boxes. The single guide RNA (sgRNA) for the CRISPR-Cas9 system and its protospacer adjacent motif (PAM) are indicated by contiguous black and red underlines, respectively. The TUBL ORF is represented by the gray shading in the box corresponding to exon 1 of TINCR . (B) Predicted secondary structure and minimal free energy for WT TINCR and the mutant form generated by the CRISPR-Cas9 system for establishment of Tubl −/− mice. The triangle indicates the 5’ end of the transcript. (C) PCR analysis of genomic DNA from the tail of mice of the indicated genotypes. The PCR products were digested with EcoRI before electrophoresis. (D) RT-qPCR analysis of TINCR in the epidermis of Tubl +/+ and Tubl −/− mice. Data are means ± SD (n = 3 independent experiments). ***p

    Journal: PLoS Genetics

    Article Title: A ubiquitin-like protein encoded by the “noncoding” RNA TINCR promotes keratinocyte proliferation and wound healing

    doi: 10.1371/journal.pgen.1009686

    Figure Lengend Snippet: Generation of TUBL-deficient mice. (A) Schematic representation of the WT TINCR allele, the single-stranded oligodeoxynucleotide (ssODN), and the mutant allele after homologous recombination. Exons are denoted by numbered boxes. The single guide RNA (sgRNA) for the CRISPR-Cas9 system and its protospacer adjacent motif (PAM) are indicated by contiguous black and red underlines, respectively. The TUBL ORF is represented by the gray shading in the box corresponding to exon 1 of TINCR . (B) Predicted secondary structure and minimal free energy for WT TINCR and the mutant form generated by the CRISPR-Cas9 system for establishment of Tubl −/− mice. The triangle indicates the 5’ end of the transcript. (C) PCR analysis of genomic DNA from the tail of mice of the indicated genotypes. The PCR products were digested with EcoRI before electrophoresis. (D) RT-qPCR analysis of TINCR in the epidermis of Tubl +/+ and Tubl −/− mice. Data are means ± SD (n = 3 independent experiments). ***p

    Article Snippet: The resulting cDNA was subjected to real-time PCR analysis with Luna Universal qPCR Master Mix (New England BioLabs) and specific primers in a StepOnePlus Real-Time PCR System (Applied Biosystems).

    Techniques: Mouse Assay, Mutagenesis, Homologous Recombination, CRISPR, Generated, Polymerase Chain Reaction, Electrophoresis, Quantitative RT-PCR

    MEOX2 depletion inhibits the sphere-forming and the growth ability of glioblastoma stem cells BT273 and BT379. ( a , b ) MEOX2 qRT-PCR analysis of BT273 ( a ) or BT379 ( b ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. The values were reported in relation to cells transduced with ctrl vector set as = 1 and normalized to PPP2CA mRNA expression ( n = 3; mean ± SD). ( c , d ) MEOX2 Western blot analysis of BT273 ( c ) or BT379 ( d ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. For BT273 and BT379, α-tubulin and β-actin were used as the internal loading controls, respectively. Representative images are shown. The bottom histograms show the quantification of MEOX2 in relation to α-tubulin and β-actin. ( n = 3; mean ± SD). ( e , f ) Sphere-forming assay of BT273 ( e ) and BT379 ( f ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. Histograms show the percentage of cells capable of re-forming a neurosphere seven days after dissociation ( n = 3; mean ± SD). Representative micrographs are shown. ( g , h ) Growth curves of BT273 ( g ) and BT379 ( h ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. ( n = 3; mean ± SD). Differences between two groups were assessed using unpaired Student’s t -test (two-tailed). Significance was defined as * p

    Journal: Cancers

    Article Title: MEOX2 Regulates the Growth and Survival of Glioblastoma Stem Cells by Modulating Genes of the Glycolytic Pathway and Response to Hypoxia

    doi: 10.3390/cancers14092304

    Figure Lengend Snippet: MEOX2 depletion inhibits the sphere-forming and the growth ability of glioblastoma stem cells BT273 and BT379. ( a , b ) MEOX2 qRT-PCR analysis of BT273 ( a ) or BT379 ( b ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. The values were reported in relation to cells transduced with ctrl vector set as = 1 and normalized to PPP2CA mRNA expression ( n = 3; mean ± SD). ( c , d ) MEOX2 Western blot analysis of BT273 ( c ) or BT379 ( d ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. For BT273 and BT379, α-tubulin and β-actin were used as the internal loading controls, respectively. Representative images are shown. The bottom histograms show the quantification of MEOX2 in relation to α-tubulin and β-actin. ( n = 3; mean ± SD). ( e , f ) Sphere-forming assay of BT273 ( e ) and BT379 ( f ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. Histograms show the percentage of cells capable of re-forming a neurosphere seven days after dissociation ( n = 3; mean ± SD). Representative micrographs are shown. ( g , h ) Growth curves of BT273 ( g ) and BT379 ( h ) cells transduced with shRNA18 or shRNA53 or ctrl lentiviral constructs. ( n = 3; mean ± SD). Differences between two groups were assessed using unpaired Student’s t -test (two-tailed). Significance was defined as * p

    Article Snippet: The resulting cDNA (25 ng) was used for the Real-time qPCR analysis using the Luna® Universal qPCR Master Mix (New England Biolabs, NEB, Ipswich, MA, USA) on a StepOnePlus instrument (Applied Biosystem, Waltham, MA, USA) according to the protocol provided by the manufacturer.

    Techniques: Quantitative RT-PCR, Transduction, Construct, Plasmid Preparation, Expressing, Western Blot, Two Tailed Test

    a) Distribution of annotated single hits over MEG3 gene, with statistically filtered EZH2-FLASH reads from two biological replicates in HUVECs. b) The occupancy of EZH2 hits over MEG3 features. Total reads per feature are given with exons being mostly occupies vs introns. c) Proportion of overlapping features over MEG3. The occupancy of EZH2 over each MEG3 exon is shown for two constitutively expressed transcripts. For both given transcripts there is high occupancy of exon 3. d) RNA immunoprecipitation (RIP) for EZH2 and H3K27me3 (repressive chromatin) followed by qPCR analysis. RIP-purified RNA from UV crosslinked HUVECs was used to prepare cDNA for qPCR analysis with primers against MEG3 (exon 3 region). Primers against U1snRNA gene serves as a negative control. Side diagram of EHZ2-MEG3 interacting region is charted as per FLASH hits and sequence. e) Distribution of EZH2 hybrids hits over MEG3 gene. Intermolecular MEG3-RNA interactions found in chimeras are captured by EZH2-FLASH-seq. Hits represent MEG3:MEG3 hybrids (black). IgG hybrids are plotted but are

    Journal: bioRxiv

    Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

    doi: 10.1101/2022.05.20.492787

    Figure Lengend Snippet: a) Distribution of annotated single hits over MEG3 gene, with statistically filtered EZH2-FLASH reads from two biological replicates in HUVECs. b) The occupancy of EZH2 hits over MEG3 features. Total reads per feature are given with exons being mostly occupies vs introns. c) Proportion of overlapping features over MEG3. The occupancy of EZH2 over each MEG3 exon is shown for two constitutively expressed transcripts. For both given transcripts there is high occupancy of exon 3. d) RNA immunoprecipitation (RIP) for EZH2 and H3K27me3 (repressive chromatin) followed by qPCR analysis. RIP-purified RNA from UV crosslinked HUVECs was used to prepare cDNA for qPCR analysis with primers against MEG3 (exon 3 region). Primers against U1snRNA gene serves as a negative control. Side diagram of EHZ2-MEG3 interacting region is charted as per FLASH hits and sequence. e) Distribution of EZH2 hybrids hits over MEG3 gene. Intermolecular MEG3-RNA interactions found in chimeras are captured by EZH2-FLASH-seq. Hits represent MEG3:MEG3 hybrids (black). IgG hybrids are plotted but are

    Article Snippet: Luna qPCR SYBR Master Mix (NEB, M3003) was used along with specific primers to determine the expression of MEG3 and housekeeping genes using Applied Biosystems QuantStudio 5 Real-Time PCR Detection System.

    Techniques: Immunoprecipitation, Real-time Polymerase Chain Reaction, Purification, Negative Control, Sequencing

    a. Venn diagram showing the intersection between statistically filtered FLASH data from two biological replicates of our MEG3-ChIRP-seq-data (green), de novo hg38 analysed GEO RNA-seq data from siEZH2 deficient HUVECs (GSE71164, blue), and EZH2 ChIP-seq following MEG3 KD (yellow) and FLASH-seq transcriptome following EZH2 IP (pink). b. Correlation between gene expression levels and FLASH signal. Gray, expressed RefSeq genes with reproducible FLASH signal consistently detected in RNA-seq. Blue, genes with the highest RNA-seq signals and no reproducible FLASH signal belonging to integrin cell surface interaction pathway. Red , expressed ITGA4 gene, and green, ITGB1 gene, without reproducible FLASH signals. Data are from two biological replicates of each EZH2 FLASH sample and three biological replicates of EZH2 RNA-seq samples (Scr vs. siEZH2, GSE71164). c. Genomic tracks showing ChIRP-seq signal (MEG3 Odd, Even and LacZ) in HUVECs over ITGA4 gene only. The MEG3 binding site is located upstream of the ITGA4 gene in the promoter region, and it overlaps with the H3K27me3 signal and EZH2; as well as downstream within the ITGA4 gene body, where it overlaps with within the EZH2 signal in the intronic region of the gene. d. MEG3-ChIRP followed by qPCR, analysis of MEG3 binding region on ITGA4 in HUVECs. The crosslinked cell lysates were incubated with combined biotinylated probes against MEG3 lncRNA and the binding complexes recovered by magnetic streptavidin-conjugated beads. The qPCR was performed to detect the enrichment of specific region that associated with MEG3, peaks were related to input control and compared vs. the non-biotynilated control. e. ChIP-QPCR enrichment for EZH2 and H3K27me3 over ITGA4 promoter region in HUVECs depleted of MEG3 vs. Control.

    Journal: bioRxiv

    Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

    doi: 10.1101/2022.05.20.492787

    Figure Lengend Snippet: a. Venn diagram showing the intersection between statistically filtered FLASH data from two biological replicates of our MEG3-ChIRP-seq-data (green), de novo hg38 analysed GEO RNA-seq data from siEZH2 deficient HUVECs (GSE71164, blue), and EZH2 ChIP-seq following MEG3 KD (yellow) and FLASH-seq transcriptome following EZH2 IP (pink). b. Correlation between gene expression levels and FLASH signal. Gray, expressed RefSeq genes with reproducible FLASH signal consistently detected in RNA-seq. Blue, genes with the highest RNA-seq signals and no reproducible FLASH signal belonging to integrin cell surface interaction pathway. Red , expressed ITGA4 gene, and green, ITGB1 gene, without reproducible FLASH signals. Data are from two biological replicates of each EZH2 FLASH sample and three biological replicates of EZH2 RNA-seq samples (Scr vs. siEZH2, GSE71164). c. Genomic tracks showing ChIRP-seq signal (MEG3 Odd, Even and LacZ) in HUVECs over ITGA4 gene only. The MEG3 binding site is located upstream of the ITGA4 gene in the promoter region, and it overlaps with the H3K27me3 signal and EZH2; as well as downstream within the ITGA4 gene body, where it overlaps with within the EZH2 signal in the intronic region of the gene. d. MEG3-ChIRP followed by qPCR, analysis of MEG3 binding region on ITGA4 in HUVECs. The crosslinked cell lysates were incubated with combined biotinylated probes against MEG3 lncRNA and the binding complexes recovered by magnetic streptavidin-conjugated beads. The qPCR was performed to detect the enrichment of specific region that associated with MEG3, peaks were related to input control and compared vs. the non-biotynilated control. e. ChIP-QPCR enrichment for EZH2 and H3K27me3 over ITGA4 promoter region in HUVECs depleted of MEG3 vs. Control.

    Article Snippet: Luna qPCR SYBR Master Mix (NEB, M3003) was used along with specific primers to determine the expression of MEG3 and housekeeping genes using Applied Biosystems QuantStudio 5 Real-Time PCR Detection System.

    Techniques: RNA Sequencing Assay, Chromatin Immunoprecipitation, Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Incubation

    a. ChIP signal enrichment vs . 1% input for EZH2 and H3K27me3 mark over ITGA4 promoter regions in HUVECs treated with A-395 (5µM, 24h) inhibitor of PRC2 vs. Control (DMSO). The expression was measured using two sets of primers against the same promoter region of ITGA4. Representative graphs are average of three qPCR datasets ± SEM. b. ITGA4 expression in the presence of A-395 vs . DMSO control, N=6 independent experiments compared using t -test. c. Measuring the expression levels of ITGA4 upon depletion of MEG3 using LNA GapmeRs (10nM, 48h), data is mean of N=5 independent experiments (biological replicates). d. Representative image of immunofluorescence staining for ITGA4 protein levels in ECs treated with A-395 vs . DMSO, or upon MEG3 depletion like in b . e. Intra-cellular localisation of MEG3 (chromatin associated lncRNA) between different cellular compartments in HUVECs treated with A-395 vs. DMSO, whereby the distribution of MEG3 has shifted upon PRC2 inhibition with A-395; from the nucleus (where it was highly chromatin bound) into the cytoplasm. Representative bars were compared by t-test and on-way Anova. f. MEG3-ChIRP followed by qPCR, N =3, analysis of MEG3 binding over ITGA4 promoter region in HUVECs treated with A-395 (5µM, 24h) vs. DMSO. MEG3-ChIRP HUVEC lysates treated with A-395 resulted in reduced engagement of MEG3 with ITGA4 site compared with either DMSO control or ChIRP with non-biotinylated probes. The non-biotin probes served as a negative control, and we detected the background level

    Journal: bioRxiv

    Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

    doi: 10.1101/2022.05.20.492787

    Figure Lengend Snippet: a. ChIP signal enrichment vs . 1% input for EZH2 and H3K27me3 mark over ITGA4 promoter regions in HUVECs treated with A-395 (5µM, 24h) inhibitor of PRC2 vs. Control (DMSO). The expression was measured using two sets of primers against the same promoter region of ITGA4. Representative graphs are average of three qPCR datasets ± SEM. b. ITGA4 expression in the presence of A-395 vs . DMSO control, N=6 independent experiments compared using t -test. c. Measuring the expression levels of ITGA4 upon depletion of MEG3 using LNA GapmeRs (10nM, 48h), data is mean of N=5 independent experiments (biological replicates). d. Representative image of immunofluorescence staining for ITGA4 protein levels in ECs treated with A-395 vs . DMSO, or upon MEG3 depletion like in b . e. Intra-cellular localisation of MEG3 (chromatin associated lncRNA) between different cellular compartments in HUVECs treated with A-395 vs. DMSO, whereby the distribution of MEG3 has shifted upon PRC2 inhibition with A-395; from the nucleus (where it was highly chromatin bound) into the cytoplasm. Representative bars were compared by t-test and on-way Anova. f. MEG3-ChIRP followed by qPCR, N =3, analysis of MEG3 binding over ITGA4 promoter region in HUVECs treated with A-395 (5µM, 24h) vs. DMSO. MEG3-ChIRP HUVEC lysates treated with A-395 resulted in reduced engagement of MEG3 with ITGA4 site compared with either DMSO control or ChIRP with non-biotinylated probes. The non-biotin probes served as a negative control, and we detected the background level

    Article Snippet: Luna qPCR SYBR Master Mix (NEB, M3003) was used along with specific primers to determine the expression of MEG3 and housekeeping genes using Applied Biosystems QuantStudio 5 Real-Time PCR Detection System.

    Techniques: Chromatin Immunoprecipitation, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Inhibition, Binding Assay, Negative Control

    a) Overview of the design of probes against MEG3 gene that were divided in probe Set1 and Set 2. The biotynilated probes were of 20 nucleotides and were spaced out 200 nucleotides apart down the gene length. b) Validation of MEG3 probes specifically binding MEG3 gene, by ChIRP-qPCR in HUVECs. Pull down with probe set 1 or set 2 retrieved 100% and 40% RNA, respectively. GAPDH primers were used as control and MEG3-associated samples did not amplify. c) Computational analysis pipeline for ChIRP-seq outlining data processing. The peak coverage was within the 100bp window. d) MEG3-ChIRP peaks associated with EZH2 gene as precipitated using both sets of probes (set 1 and 2). e) Enrichment of MEG3 signal by ChIRP-qpcr versus negative control (Background) at named promoter regions. MEG3 binding to genomic loci as validate by ChIRP-qPCR in HUVECs. Pull downs were performed with joined Odd and Even probes. Value 1 is a background level, defined by enrichment to LacZ negative probes in ChIRP. Control primers were designed for positive ChIRP peaks and used as a positive control and for regions deprived of MEG3-ChIRP reads as a negative control .

    Journal: bioRxiv

    Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

    doi: 10.1101/2022.05.20.492787

    Figure Lengend Snippet: a) Overview of the design of probes against MEG3 gene that were divided in probe Set1 and Set 2. The biotynilated probes were of 20 nucleotides and were spaced out 200 nucleotides apart down the gene length. b) Validation of MEG3 probes specifically binding MEG3 gene, by ChIRP-qPCR in HUVECs. Pull down with probe set 1 or set 2 retrieved 100% and 40% RNA, respectively. GAPDH primers were used as control and MEG3-associated samples did not amplify. c) Computational analysis pipeline for ChIRP-seq outlining data processing. The peak coverage was within the 100bp window. d) MEG3-ChIRP peaks associated with EZH2 gene as precipitated using both sets of probes (set 1 and 2). e) Enrichment of MEG3 signal by ChIRP-qpcr versus negative control (Background) at named promoter regions. MEG3 binding to genomic loci as validate by ChIRP-qPCR in HUVECs. Pull downs were performed with joined Odd and Even probes. Value 1 is a background level, defined by enrichment to LacZ negative probes in ChIRP. Control primers were designed for positive ChIRP peaks and used as a positive control and for regions deprived of MEG3-ChIRP reads as a negative control .

    Article Snippet: Luna qPCR SYBR Master Mix (NEB, M3003) was used along with specific primers to determine the expression of MEG3 and housekeeping genes using Applied Biosystems QuantStudio 5 Real-Time PCR Detection System.

    Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Negative Control, Positive Control

    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