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 1 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 "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

    5) 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

    6) 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

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) Product Images from "Evaluation Of SYBR Green Real Time PCR For Detecting SARS-CoV-2 From Clinical Samples"

    Article Title: Evaluation Of SYBR Green Real Time PCR For Detecting SARS-CoV-2 From Clinical Samples

    Journal: bioRxiv

    doi: 10.1101/2020.05.13.093609

    Real time PCR results, from SYBER and Taqman chemistries, of different dilutions of the control vectors for the targeted regions: ORF1b-nsp14 and N (left and right panels, respectively). A) and B) show the amplification plots for the RT-qPCR protocol employing fluorogenic probes. C) and D) show the amplification plots for the qPCR protocol developed in this study employing SYBR Green as a nucleic acid dye. E) and F) show the melting curves for the products amplified with the SYBR Green-based qPCR protocol. Below these panels are the references for each of the dilutions assayed expressed in plasmid copies (C-: non-template control). G) and H) show agarose gel electrophoresis of PCR products amplified with the SYBR Green-based qPCR protocol. MW: 100bp DNA Molecular Weight (New England Biolabs); lanes 1 to 4: control dilutions (10 4 , 10 5 , 10 6 and 10 7 copies/μL, respectively); lane 5: non-template-control.
    Figure Legend Snippet: Real time PCR results, from SYBER and Taqman chemistries, of different dilutions of the control vectors for the targeted regions: ORF1b-nsp14 and N (left and right panels, respectively). A) and B) show the amplification plots for the RT-qPCR protocol employing fluorogenic probes. C) and D) show the amplification plots for the qPCR protocol developed in this study employing SYBR Green as a nucleic acid dye. E) and F) show the melting curves for the products amplified with the SYBR Green-based qPCR protocol. Below these panels are the references for each of the dilutions assayed expressed in plasmid copies (C-: non-template control). G) and H) show agarose gel electrophoresis of PCR products amplified with the SYBR Green-based qPCR protocol. MW: 100bp DNA Molecular Weight (New England Biolabs); lanes 1 to 4: control dilutions (10 4 , 10 5 , 10 6 and 10 7 copies/μL, respectively); lane 5: non-template-control.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, SYBR Green Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Molecular Weight

    13) Product Images from "The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function"

    Article Title: The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function

    Journal: PeerJ

    doi: 10.7717/peerj.4093

    qPCR analysis of α-crystallin expression in zebrafish embryos. Box and whisker plot shows delta Ct for the three zebrafish α-crystallins, indicating mRNA levels relative to three endogenous controls from 12 h post fertilization (0.5 dpf) to 5 dpf. Lower numerical Ct values on these inverted y -axes indicate increased expression. All three graphs show low initial baseline expression that increases in αA (A) and αBa-crystallin (B), but stays consistently low in αBb-crystallin (C). Alpha A-crystallin expression increased earlier than αBa-crystallin expression. Asterisks indicate statistically significant differences in expression compared to the 0.5 dpf timepoint ( p
    Figure Legend Snippet: qPCR analysis of α-crystallin expression in zebrafish embryos. Box and whisker plot shows delta Ct for the three zebrafish α-crystallins, indicating mRNA levels relative to three endogenous controls from 12 h post fertilization (0.5 dpf) to 5 dpf. Lower numerical Ct values on these inverted y -axes indicate increased expression. All three graphs show low initial baseline expression that increases in αA (A) and αBa-crystallin (B), but stays consistently low in αBb-crystallin (C). Alpha A-crystallin expression increased earlier than αBa-crystallin expression. Asterisks indicate statistically significant differences in expression compared to the 0.5 dpf timepoint ( p

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

    14) Product Images from "Lysophosphatidylcholine-induced mitochondrial fission contributes to collagen production in human cardiac fibroblasts"

    Article Title: Lysophosphatidylcholine-induced mitochondrial fission contributes to collagen production in human cardiac fibroblasts

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.RA119000141

    LPC-induced COX-2 expression is mediated via a mitoROS-PKCα-Drp1-JNK1/2-dependent FoxO1 pathway. A: HCFs were treated with various concentrations of AS1842856 for 1 h prior to LPC treatment for 6 h. Western blot analyses were performed to determine the levels of COX-2 and GAPDH. Densitometry analyses of COX-2 protein levels were normalized to GAPDH and relative to control (0.5% DMSO and 0.5% EtOH), n = 5. B: HCFs were pretreated with AS1842856 for 1 h, and then treated with LPC for 4 h. RT-qPCR was performed to measure the COX-2 and GAPDH gene expression (open bars), n = 6. A dual luciferase activity assay was conducted to determine COX-2 promoter activity (gray bars), n = 5. Values are relative to control (0.5% DMSO and 0.5% EtOH). C: HCFs were pretreated with MitoTEMPO (1 μM), Gö 6976 (100 nM), mdivi-1 (50 nM), SP600125 (1 μM), or AS1842856 (100 nM) for 1 h followed LPC (40 μM) treatment for 1 h. The nuclear fraction was prepared and subjected to Western blot analysis using an anti-FoxO1, anti-phospho-FoxO1 S256 , or anti-lamin A (as a marker protein for nuclear fractions) antibody. Densitometry analyses of phospho-FoxO1 S256 levels were normalized to lamin A and relative to control (0.5% DMSO and 0.5% EtOH), n = 5. D: HCFs were pretreated with MitoTEMPO (1 μM), Gö 6976 (100 nM), mdivi-1 (50 nM), dynasore (300 nM), SP600125 (1 μM), or AS1842856 (100 nM) and incubated with LPC for 1 h. The DNA binding activity of FoxO1 was determined by a chromatin immunoprecipitation assay using anti-phospho-FoxO1 S256 antibody. The enrichment of specific DNA or input DNA was determined by qPCR analysis. Results are relative to control (0.5% DMSO and 0.5% EtOH) after being normalized to input control, n = 5. Data are expressed as the mean ± SEM, and were analyzed by one-way ANOVA with Tukey’s post hoc tests. *P
    Figure Legend Snippet: LPC-induced COX-2 expression is mediated via a mitoROS-PKCα-Drp1-JNK1/2-dependent FoxO1 pathway. A: HCFs were treated with various concentrations of AS1842856 for 1 h prior to LPC treatment for 6 h. Western blot analyses were performed to determine the levels of COX-2 and GAPDH. Densitometry analyses of COX-2 protein levels were normalized to GAPDH and relative to control (0.5% DMSO and 0.5% EtOH), n = 5. B: HCFs were pretreated with AS1842856 for 1 h, and then treated with LPC for 4 h. RT-qPCR was performed to measure the COX-2 and GAPDH gene expression (open bars), n = 6. A dual luciferase activity assay was conducted to determine COX-2 promoter activity (gray bars), n = 5. Values are relative to control (0.5% DMSO and 0.5% EtOH). C: HCFs were pretreated with MitoTEMPO (1 μM), Gö 6976 (100 nM), mdivi-1 (50 nM), SP600125 (1 μM), or AS1842856 (100 nM) for 1 h followed LPC (40 μM) treatment for 1 h. The nuclear fraction was prepared and subjected to Western blot analysis using an anti-FoxO1, anti-phospho-FoxO1 S256 , or anti-lamin A (as a marker protein for nuclear fractions) antibody. Densitometry analyses of phospho-FoxO1 S256 levels were normalized to lamin A and relative to control (0.5% DMSO and 0.5% EtOH), n = 5. D: HCFs were pretreated with MitoTEMPO (1 μM), Gö 6976 (100 nM), mdivi-1 (50 nM), dynasore (300 nM), SP600125 (1 μM), or AS1842856 (100 nM) and incubated with LPC for 1 h. The DNA binding activity of FoxO1 was determined by a chromatin immunoprecipitation assay using anti-phospho-FoxO1 S256 antibody. The enrichment of specific DNA or input DNA was determined by qPCR analysis. Results are relative to control (0.5% DMSO and 0.5% EtOH) after being normalized to input control, n = 5. Data are expressed as the mean ± SEM, and were analyzed by one-way ANOVA with Tukey’s post hoc tests. *P

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Luciferase, Activity Assay, Marker, Incubation, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    LPC-induced COX-2-dependent collagen secretion is mediated via EP 4 receptors. A: HCFs were pretreated with MitoTEMPO (1 μM), Gö 6976 (100 nM), mdivi-1 (50 nM), dynasore (300 nM), SP600125 (1 μM), AS1842856 (100 nM), celecoxib (10 μM), or NS-398 (10 μM) for 1 h and then incubated with LPC (40 μM) for 6 h. The conditioned media were subjected to determination of PGE 2 production, n = 7. B: HCFs were transfected with siRNA of EP 2 , EP 3 , or EP 4 and scrambled sequences, and then incubated with LPC for 48 h. RT-qPCR was performed to determine the EPs gene expression as shown in supplemental Fig. S6B. A Sircol collagen assay was conducted to determine levels of collagen, n = 6. Results are relative to control (0.5% EtOH). Values are expressed as the mean ± SEM and were analyzed using one-way ANOVA with Tukey’s post hoc tests. *P
    Figure Legend Snippet: LPC-induced COX-2-dependent collagen secretion is mediated via EP 4 receptors. A: HCFs were pretreated with MitoTEMPO (1 μM), Gö 6976 (100 nM), mdivi-1 (50 nM), dynasore (300 nM), SP600125 (1 μM), AS1842856 (100 nM), celecoxib (10 μM), or NS-398 (10 μM) for 1 h and then incubated with LPC (40 μM) for 6 h. The conditioned media were subjected to determination of PGE 2 production, n = 7. B: HCFs were transfected with siRNA of EP 2 , EP 3 , or EP 4 and scrambled sequences, and then incubated with LPC for 48 h. RT-qPCR was performed to determine the EPs gene expression as shown in supplemental Fig. S6B. A Sircol collagen assay was conducted to determine levels of collagen, n = 6. Results are relative to control (0.5% EtOH). Values are expressed as the mean ± SEM and were analyzed using one-way ANOVA with Tukey’s post hoc tests. *P

    Techniques Used: Incubation, Transfection, Quantitative RT-PCR, Expressing, Sircol Collagen Assay

    LPC-induced mitoROS generation is involved in COX-2-dependent collagen secretion. A: HCFs were pretreated with either MitoTEMPO (1 μM) or MitoQ (100 nM) for 1 h, and then treated with either 0.5% EtOH (vehicle control) or LPC (40 μM) for the indicated time intervals (0, 15, 30, 60 min). mitoROS generation was detected under a fluorescence microscope with MitoSOX Red. Representative images are shown. Scale bar indicates 100 μm, n = 5. B: HCFs were pretreated with either MitoTEMPO or MitoQ for 1 h, and then incubated with either 0.5% EtOH (vehicle control) or LPC for the indicated time intervals. mitoROS production was determined by MitoSOX Red staining. The fluorescence unit of MitoSOX Red was measured using a fluorescent microplate reader. Values are shown as fold change, n = 5. C, D: HCFs were pretreated with either MitoTEMPO or MitoQ for 1 h followed by LPC for 6 h. The protein levels of COX-2 and GAPDH were determined by Western blotting. Densitometry analyses of COX-2 protein levels were normalized to GAPDH and relative to control (0.5% DMSO and 0.5% EtOH), n = 5. E: HCFs were pretreated with either MitoTEMPO or MitoQ for 1 h followed by LPC for 4 h. The mRNA levels of COX-2 and GAPDH were determined by using RT-qPCR (open bars). Results were normalized to GAPDH and relative to control (0.5% DMSO and 0.5% EtOH), n = 6. HCFs were cotransfected with pGL3b-cox-2-luc and pCMV-β-gal plasmid for 24 h and pretreated with either MitoTEMPO or MitoQ for 1 h followed by LPC for 4 h. COX-2 promoter activity was evaluated with a dual luciferase activity assay (gray bars). Results were normalized to β-gal activity and relative to control (0.5% DMSO and 0.5% EtOH), n = 6. F, G: Mouse heart segments were pretreated with MitoTEMPO for 1 h followed LPC treatment for 6 h in Krebs solution bubbled with 5% O 2 and 95% CO 2 at 37°C. F: The heart tissues were homogenized and subjected to glutathione detection assay. The oxidative stress of heart was reported as the ratio of GSH to GSSG, n = 5. G: Western blot analysis or RT-qPCR were performed to determine the levels of COX-2 protein (open bars; n = 5) and mRNA (gray bars; n = 5). Values were relative to control (0.5% DMSO and 0.5% EtOH). H: HCFs were pretreated with either celecoxib (3 μM), NS-398 (1 μM), or MitoTEMPO (1 μM) followed by LPC or PGE 2 for the indicated time points. The conditioned media were subjected to Sircol collagen assay, n = 5. Data are presented as mean ± SEM and were analyzed by one-way ANOVA with Tukey’s post hoc tests. *P
    Figure Legend Snippet: LPC-induced mitoROS generation is involved in COX-2-dependent collagen secretion. A: HCFs were pretreated with either MitoTEMPO (1 μM) or MitoQ (100 nM) for 1 h, and then treated with either 0.5% EtOH (vehicle control) or LPC (40 μM) for the indicated time intervals (0, 15, 30, 60 min). mitoROS generation was detected under a fluorescence microscope with MitoSOX Red. Representative images are shown. Scale bar indicates 100 μm, n = 5. B: HCFs were pretreated with either MitoTEMPO or MitoQ for 1 h, and then incubated with either 0.5% EtOH (vehicle control) or LPC for the indicated time intervals. mitoROS production was determined by MitoSOX Red staining. The fluorescence unit of MitoSOX Red was measured using a fluorescent microplate reader. Values are shown as fold change, n = 5. C, D: HCFs were pretreated with either MitoTEMPO or MitoQ for 1 h followed by LPC for 6 h. The protein levels of COX-2 and GAPDH were determined by Western blotting. Densitometry analyses of COX-2 protein levels were normalized to GAPDH and relative to control (0.5% DMSO and 0.5% EtOH), n = 5. E: HCFs were pretreated with either MitoTEMPO or MitoQ for 1 h followed by LPC for 4 h. The mRNA levels of COX-2 and GAPDH were determined by using RT-qPCR (open bars). Results were normalized to GAPDH and relative to control (0.5% DMSO and 0.5% EtOH), n = 6. HCFs were cotransfected with pGL3b-cox-2-luc and pCMV-β-gal plasmid for 24 h and pretreated with either MitoTEMPO or MitoQ for 1 h followed by LPC for 4 h. COX-2 promoter activity was evaluated with a dual luciferase activity assay (gray bars). Results were normalized to β-gal activity and relative to control (0.5% DMSO and 0.5% EtOH), n = 6. F, G: Mouse heart segments were pretreated with MitoTEMPO for 1 h followed LPC treatment for 6 h in Krebs solution bubbled with 5% O 2 and 95% CO 2 at 37°C. F: The heart tissues were homogenized and subjected to glutathione detection assay. The oxidative stress of heart was reported as the ratio of GSH to GSSG, n = 5. G: Western blot analysis or RT-qPCR were performed to determine the levels of COX-2 protein (open bars; n = 5) and mRNA (gray bars; n = 5). Values were relative to control (0.5% DMSO and 0.5% EtOH). H: HCFs were pretreated with either celecoxib (3 μM), NS-398 (1 μM), or MitoTEMPO (1 μM) followed by LPC or PGE 2 for the indicated time points. The conditioned media were subjected to Sircol collagen assay, n = 5. Data are presented as mean ± SEM and were analyzed by one-way ANOVA with Tukey’s post hoc tests. *P

    Techniques Used: Fluorescence, Microscopy, Incubation, Staining, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Activity Assay, Luciferase, Detection Assay, Sircol Collagen Assay

    Drp1 is involved in mitochondrial fission and depolarization and COX-2 expression induced by LPC. A: HCFs were treated with LPC (40 μM) for the indicated time intervals. Immunofluorescence staining was performed with anti-phospho-Drp1 S616 and anti-TOM20 antibodies, labeled with FITC (green) and rhodamine (red)-conjugated IgG, respectively. DNA was counterstained with DAPI (blue). Arrows indicate the localization of TOM20 and phospho-Drp1 S616 . Representative fluorescence images are presented. Scale bar indicates 100 μm, n = 5. Quantification of the fluorescence intensity of phospho-Drp1 S616 is presented in supplemental Fig. S1B. B, C: HCFs were pretreated with mdivi-1 or dynasore for 1 h prior to LPC treatment for 6 h. Western blot analyses were performed to measure the levels of COX-2 and GAPDH protein. Densitometry analysis of COX-2 was normalized to GAPDH and relative to control (0.5% DMSO and 0.5% EtOH), n = 5. D: HCFs were pretreated with mdivi-1 (50 nM) or dynasore (300 nM) for 1 h, and then treated with LPC for 4 h. RT-qPCR was performed to measure COX-2 expression (open bars), n = 5. A dual luciferase activity assay was conducted to evaluate COX-2 promoter activity (gray bars), n = 5. Values are relative to control (0.5% DMSO and 0.5% EtOH). E: The conditioned media were subjected to determination of collagen contents under pretreatment with mdivi-1 (50 nM) or dynasore (300 nM) for 1 h, and then challenged with LPC for 48 h in HCFs, n = 5. Data are presented as the mean ± SEM, and were analyzed by one-way ANOVA with Tukey’s post hoc tests. *P
    Figure Legend Snippet: Drp1 is involved in mitochondrial fission and depolarization and COX-2 expression induced by LPC. A: HCFs were treated with LPC (40 μM) for the indicated time intervals. Immunofluorescence staining was performed with anti-phospho-Drp1 S616 and anti-TOM20 antibodies, labeled with FITC (green) and rhodamine (red)-conjugated IgG, respectively. DNA was counterstained with DAPI (blue). Arrows indicate the localization of TOM20 and phospho-Drp1 S616 . Representative fluorescence images are presented. Scale bar indicates 100 μm, n = 5. Quantification of the fluorescence intensity of phospho-Drp1 S616 is presented in supplemental Fig. S1B. B, C: HCFs were pretreated with mdivi-1 or dynasore for 1 h prior to LPC treatment for 6 h. Western blot analyses were performed to measure the levels of COX-2 and GAPDH protein. Densitometry analysis of COX-2 was normalized to GAPDH and relative to control (0.5% DMSO and 0.5% EtOH), n = 5. D: HCFs were pretreated with mdivi-1 (50 nM) or dynasore (300 nM) for 1 h, and then treated with LPC for 4 h. RT-qPCR was performed to measure COX-2 expression (open bars), n = 5. A dual luciferase activity assay was conducted to evaluate COX-2 promoter activity (gray bars), n = 5. Values are relative to control (0.5% DMSO and 0.5% EtOH). E: The conditioned media were subjected to determination of collagen contents under pretreatment with mdivi-1 (50 nM) or dynasore (300 nM) for 1 h, and then challenged with LPC for 48 h in HCFs, n = 5. Data are presented as the mean ± SEM, and were analyzed by one-way ANOVA with Tukey’s post hoc tests. *P

    Techniques Used: Expressing, Immunofluorescence, Staining, Labeling, Fluorescence, Western Blot, Quantitative RT-PCR, Luciferase, Activity Assay

    mitoROS/PKCα/JNK is involved in LPC-induced COX-2 expression and collagen secretion. A, B: HCFs were pretreated with various concentrations of Gö 6976 or Gö 6983 for 1 h, and then incubated with LPC for 6 h. The protein levels of COX-2 and GAPDH were determined by Western blot analysis. Densitometry analyses of COX-2 protein levels were normalized to GAPDH and relative to control (0.5% DMSO and 0.5% EtOH), n = 6. C: HCFs were pretreated with Gö 6976 (100 nM), Gö 6983 (30 nM), or SP600125 (1 μM) for 1 h, and then treated with LPC for 4 h. RT-qPCR was performed to measure COX-2 expression (open bars), n = 6. A dual luciferase activity assay was conducted to evaluate COX-2 promoter activity (gray bars), n = 5. Values are relative to control (0.5% DMSO and 0.5% EtOH). D: HCFs were pretreated with either Gö 6976 (100 nM) or SP600125 (1 μM) for 1 h followed by LPC treatment for 48 h. The conditioned media were subjected to evaluation of collagen content, n = 5. E, F: HCFs were pretreated with mdivi-1 (50 nM), Gö 6976 (100 nM), or SP600125 (1 μM) for 1 h, and then treated with LPC (40 μM) for the indicated time points. E: mitoROS production was determined by MitoSOX Red staining. The fluorescence unit of MitoSOX Red was measured using a fluorescent microplate reader. Results are relative to control, n = 7. F: The protein levels were determined by Western blotting. The densitometry measurements are presented in supplemental Fig. S2A, n = 7. Data are expressed as the mean ± SEM and were analyzed by one-way ANOVA with Tukey’s post hoc tests. *P
    Figure Legend Snippet: mitoROS/PKCα/JNK is involved in LPC-induced COX-2 expression and collagen secretion. A, B: HCFs were pretreated with various concentrations of Gö 6976 or Gö 6983 for 1 h, and then incubated with LPC for 6 h. The protein levels of COX-2 and GAPDH were determined by Western blot analysis. Densitometry analyses of COX-2 protein levels were normalized to GAPDH and relative to control (0.5% DMSO and 0.5% EtOH), n = 6. C: HCFs were pretreated with Gö 6976 (100 nM), Gö 6983 (30 nM), or SP600125 (1 μM) for 1 h, and then treated with LPC for 4 h. RT-qPCR was performed to measure COX-2 expression (open bars), n = 6. A dual luciferase activity assay was conducted to evaluate COX-2 promoter activity (gray bars), n = 5. Values are relative to control (0.5% DMSO and 0.5% EtOH). D: HCFs were pretreated with either Gö 6976 (100 nM) or SP600125 (1 μM) for 1 h followed by LPC treatment for 48 h. The conditioned media were subjected to evaluation of collagen content, n = 5. E, F: HCFs were pretreated with mdivi-1 (50 nM), Gö 6976 (100 nM), or SP600125 (1 μM) for 1 h, and then treated with LPC (40 μM) for the indicated time points. E: mitoROS production was determined by MitoSOX Red staining. The fluorescence unit of MitoSOX Red was measured using a fluorescent microplate reader. Results are relative to control, n = 7. F: The protein levels were determined by Western blotting. The densitometry measurements are presented in supplemental Fig. S2A, n = 7. Data are expressed as the mean ± SEM and were analyzed by one-way ANOVA with Tukey’s post hoc tests. *P

    Techniques Used: Expressing, Incubation, Western Blot, Quantitative RT-PCR, Luciferase, Activity Assay, Staining, Fluorescence

    15) Product Images from "Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies"

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    Journal: bioRxiv

    doi: 10.1101/2021.04.03.438258

    ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.
    Figure Legend Snippet: ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.

    Techniques Used: Over Expression, Activity Assay, Neutralization, Infection, Purification, Labeling, Protein Binding, Flow Cytometry, Quantitative RT-PCR, Luciferase

    16) Product Images from "Novel mosaic mice with diverse applications"

    Article Title: Novel mosaic mice with diverse applications

    Journal: bioRxiv

    doi: 10.1101/2020.03.21.001388

    Creation and characterization of MARC-11. A-B) Generation of MARC-11. The 11-gRNA array contains a single CD19 gRNA gene, 5 CD47 gRNA genes (CD47.1 to CD47.5) and 5 CD4 gRNA genes (CD4.1 to CD4.5; Fig. 2A , top). The transgene was inserted into the H11 locus ( Fig. 2A , bottom). The thick blue lines flanking the transgene are the homology arms in the targeting vector. F1/R1 and F2/R2 are PCR primers for screening the founders ( Fig. 2B ). C) Quantification of CD19 expression by FACS (left), of indels at the CD19 locus by PCR-Sanger sequencing (middle) and of the CD19 gRNA expression by qRT-PCR (right). Values from the qRT-PCR are mean+/-SD. D) Transgene recombination as revealed by PCR detecting all forms of transgenes including intact transgene and “zero-mer”. PCR primer pair is F/R (F/R’ was used instead for experiments in Fig. 2E ). The pink arrows indicate a nonspecific amplicon that only appeared in the absence of recombination and migrated slightly faster than the monomer. For unknown reasons, residual amounts of intact array tended to persist even when dimers were fully undetectable. Samples were from F6 mice (counting the founders as F1). E) Total abundance of various P0 gRNA genes as a whole in TAM treated mice relative to that in untreated mice. Thymocytes were analyzed on Day 8 following TAM. The primer pair used (F/R’) are depicted in Fig. 2D , with the R’ binding the scaffold common to all gRNA genes. The qPCR signals were normalized to an internal control ( Brg1 ) and plotted relative to that of the samples lacking TAM treatment. F) Representation of individual gRNA genes at P0 following recombination. Samples were harvested on Day 8 following TAM (80 ug/g). PCR was performed using a primer pair located at similar positions to F/R’ depicted in Fig. 2D (F3/R3, not shown), and the P0 gRNA gene identified by NGS. The representation of gRNA genes at P0 in various organs was shown at the top, whereas the bottom plot shows the P0 gRNA gene representation in the CD4 + subset of the TCR - CD8 + thymocytes (TCR - D4 + CD8 + ) relative to the unfractionated counterpart (TCR-CD8 + ; see Fig. 2G for the two populations). G) FACS analysis of CD4 and CD47 expression in thymocytes. Cells were stained with CD4, CD8, TCR and CD47 antibodies before the subsets that should normally express CD4 (namely, the cells at the DP and CD4 cell stages, marked by TCR - D8 + and TCR+CD8 - , respectively) were analyzed for CD4 vs. CD47 expression. The red and turquoise circles mark the CD4 + subset and their unfractionated counterpart, respectively, that were used for the NGS experiment shown at the bottom of Fig. 2F .
    Figure Legend Snippet: Creation and characterization of MARC-11. A-B) Generation of MARC-11. The 11-gRNA array contains a single CD19 gRNA gene, 5 CD47 gRNA genes (CD47.1 to CD47.5) and 5 CD4 gRNA genes (CD4.1 to CD4.5; Fig. 2A , top). The transgene was inserted into the H11 locus ( Fig. 2A , bottom). The thick blue lines flanking the transgene are the homology arms in the targeting vector. F1/R1 and F2/R2 are PCR primers for screening the founders ( Fig. 2B ). C) Quantification of CD19 expression by FACS (left), of indels at the CD19 locus by PCR-Sanger sequencing (middle) and of the CD19 gRNA expression by qRT-PCR (right). Values from the qRT-PCR are mean+/-SD. D) Transgene recombination as revealed by PCR detecting all forms of transgenes including intact transgene and “zero-mer”. PCR primer pair is F/R (F/R’ was used instead for experiments in Fig. 2E ). The pink arrows indicate a nonspecific amplicon that only appeared in the absence of recombination and migrated slightly faster than the monomer. For unknown reasons, residual amounts of intact array tended to persist even when dimers were fully undetectable. Samples were from F6 mice (counting the founders as F1). E) Total abundance of various P0 gRNA genes as a whole in TAM treated mice relative to that in untreated mice. Thymocytes were analyzed on Day 8 following TAM. The primer pair used (F/R’) are depicted in Fig. 2D , with the R’ binding the scaffold common to all gRNA genes. The qPCR signals were normalized to an internal control ( Brg1 ) and plotted relative to that of the samples lacking TAM treatment. F) Representation of individual gRNA genes at P0 following recombination. Samples were harvested on Day 8 following TAM (80 ug/g). PCR was performed using a primer pair located at similar positions to F/R’ depicted in Fig. 2D (F3/R3, not shown), and the P0 gRNA gene identified by NGS. The representation of gRNA genes at P0 in various organs was shown at the top, whereas the bottom plot shows the P0 gRNA gene representation in the CD4 + subset of the TCR - CD8 + thymocytes (TCR - D4 + CD8 + ) relative to the unfractionated counterpart (TCR-CD8 + ; see Fig. 2G for the two populations). G) FACS analysis of CD4 and CD47 expression in thymocytes. Cells were stained with CD4, CD8, TCR and CD47 antibodies before the subsets that should normally express CD4 (namely, the cells at the DP and CD4 cell stages, marked by TCR - D8 + and TCR+CD8 - , respectively) were analyzed for CD4 vs. CD47 expression. The red and turquoise circles mark the CD4 + subset and their unfractionated counterpart, respectively, that were used for the NGS experiment shown at the bottom of Fig. 2F .

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Expressing, FACS, Sequencing, Quantitative RT-PCR, Amplification, Mouse Assay, Binding Assay, Real-time Polymerase Chain Reaction, Next-Generation Sequencing, Staining

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

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

    Journal: bioRxiv

    doi: 10.1101/700674

    RT-qPCR and qPCR of E. coli O157:H7 from 0, 4, 8, 24 and 72 h growth in BHI. (A) The growth curve of E. coli O157:H7 at 0, 4, 8, 24, 72 h in BHI. The initial concentration was 3-log CFU/mL. (B) RT-qPCR for RNA collected from 5 time points. (C) qPCR for 0-72 h RNA as the negative control (NC) for DNA contamination – no DNA contamination was found in those samples. (D) qPCR for DNA collected from 5 time points. (E) The melting curve analysis of RT-qPCR for 0-72 h RNA.
    Figure Legend Snippet: RT-qPCR and qPCR of E. coli O157:H7 from 0, 4, 8, 24 and 72 h growth in BHI. (A) The growth curve of E. coli O157:H7 at 0, 4, 8, 24, 72 h in BHI. The initial concentration was 3-log CFU/mL. (B) RT-qPCR for RNA collected from 5 time points. (C) qPCR for 0-72 h RNA as the negative control (NC) for DNA contamination – no DNA contamination was found in those samples. (D) qPCR for DNA collected from 5 time points. (E) The melting curve analysis of RT-qPCR for 0-72 h RNA.

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

    18) Product Images from "SMRT-Cappable-seq reveals complex operon variants in bacteria"

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria

    Journal: bioRxiv

    doi: 10.1101/262964

    The riboswitch structure controls the condition-dependent read-through across the dapA-bamC termination site. A . An example of the dapA-ypfJ operon containing the previously known dapA-bamC TTS (red arrow), where the rate of read-through is condition dependent. B . Schema of this TTS location. The Riboswitch structure (blue) that locates 10 bp upstream of the termination site (red) is deleted to examine the role of this regulatory region in the control of transcription termination. C. D . The level of read-through across the TTS of wild-type (WT) and deletion strain (DEL) were measured for bacteria grown in both M9 and Rich medium by qPCR. The qPCR1 primers amplify a upstream region of the predicted riboswitch site. For qPCR product 2, the forward primer binds to the 5’ end of the known dapA-bamC TTS while the reverse primers binds to the downstream region of the TTS. Therefore, read-through ratio was calculated as the amount of qPCR2 product divided by qPCR1 product. Data shown are the means ± SD from five independent repeats. Unpaired t-test was used to determine significance. The read-through in DEL is significantly higher than in WT in Rich condition.
    Figure Legend Snippet: The riboswitch structure controls the condition-dependent read-through across the dapA-bamC termination site. A . An example of the dapA-ypfJ operon containing the previously known dapA-bamC TTS (red arrow), where the rate of read-through is condition dependent. B . Schema of this TTS location. The Riboswitch structure (blue) that locates 10 bp upstream of the termination site (red) is deleted to examine the role of this regulatory region in the control of transcription termination. C. D . The level of read-through across the TTS of wild-type (WT) and deletion strain (DEL) were measured for bacteria grown in both M9 and Rich medium by qPCR. The qPCR1 primers amplify a upstream region of the predicted riboswitch site. For qPCR product 2, the forward primer binds to the 5’ end of the known dapA-bamC TTS while the reverse primers binds to the downstream region of the TTS. Therefore, read-through ratio was calculated as the amount of qPCR2 product divided by qPCR1 product. Data shown are the means ± SD from five independent repeats. Unpaired t-test was used to determine significance. The read-through in DEL is significantly higher than in WT in Rich condition.

    Techniques Used: Real-time Polymerase Chain Reaction

    19) Product Images from "Ex Vivo Hepatocyte Reprograming Promotes Homology‐Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation"

    Article Title: Ex Vivo Hepatocyte Reprograming Promotes Homology‐Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation

    Journal: Hepatology Communications

    doi: 10.1002/hep4.1315

    Ex vivo reprogramming of cultured hepatocytes up‐regulates DNA repair pathways. (A) Averaged relative quantification values showing expression levels of five genes involved in homologous recombination ( Brca1 , Brca2 , Rad51 , Rad52 , and Pcna ) relative to the length of culture as determined by comparative qPCR on RNA isolated from primary hepatocytes. Samples are standardized to B2m expression. (B) Deconvolution of these five genes, showing consistent up‐regulation over time. (C) Representative histogram of Ki‐67 intensity in primary hepatocytes after increasing time in culture, with peak heights normalized to mode of sample. (D) Quantification of mean fluorescence intensity in biological replicates (n = 2). (E) Hierarchical clustering of six RNA‐Seq samples at three different time points (0 hours, 24 hours, and 48 hours). The 24‐hour and 48‐hour time points are more similar to one another than either is to 0 hours. (F) Heat map of gene expression in all samples for differentially expressed genes between times 0 hours and 48 hours (n = 5,254). (G) Enrichment analysis of highly affected gene sets clustered by biologic functions between cells in culture for 48 hours relative to 0 hours. (H) Detailed enrichment summary illustrating differences in the regulation of two selected Gene Ontology function annotations of interest—DNA repair (horizontal black bars on the left) and cellular amino acid catabolic process (horizontal green bars on the left)—among the three time points collected (presented by column). The heat map includes the top and bottom 500 differentially expressed genes ranked by fold change between times 0 hours and 48 hours. Individual DNA repair genes are up‐regulated, whereas individual cellular and amino acid catabolic process genes are down‐regulated in the 24‐hour and 48‐hour groups. Abbreviations: FDR, false discovery rate; and ns, not significant at α = 0.05.
    Figure Legend Snippet: Ex vivo reprogramming of cultured hepatocytes up‐regulates DNA repair pathways. (A) Averaged relative quantification values showing expression levels of five genes involved in homologous recombination ( Brca1 , Brca2 , Rad51 , Rad52 , and Pcna ) relative to the length of culture as determined by comparative qPCR on RNA isolated from primary hepatocytes. Samples are standardized to B2m expression. (B) Deconvolution of these five genes, showing consistent up‐regulation over time. (C) Representative histogram of Ki‐67 intensity in primary hepatocytes after increasing time in culture, with peak heights normalized to mode of sample. (D) Quantification of mean fluorescence intensity in biological replicates (n = 2). (E) Hierarchical clustering of six RNA‐Seq samples at three different time points (0 hours, 24 hours, and 48 hours). The 24‐hour and 48‐hour time points are more similar to one another than either is to 0 hours. (F) Heat map of gene expression in all samples for differentially expressed genes between times 0 hours and 48 hours (n = 5,254). (G) Enrichment analysis of highly affected gene sets clustered by biologic functions between cells in culture for 48 hours relative to 0 hours. (H) Detailed enrichment summary illustrating differences in the regulation of two selected Gene Ontology function annotations of interest—DNA repair (horizontal black bars on the left) and cellular amino acid catabolic process (horizontal green bars on the left)—among the three time points collected (presented by column). The heat map includes the top and bottom 500 differentially expressed genes ranked by fold change between times 0 hours and 48 hours. Individual DNA repair genes are up‐regulated, whereas individual cellular and amino acid catabolic process genes are down‐regulated in the 24‐hour and 48‐hour groups. Abbreviations: FDR, false discovery rate; and ns, not significant at α = 0.05.

    Techniques Used: Ex Vivo, Cell Culture, Expressing, Homologous Recombination, Real-time Polymerase Chain Reaction, Isolation, Fluorescence, RNA Sequencing Assay

    20) Product Images from "Efficient Propagation of Circulating Tumor Cells: A First Step for Probing Tumor Metastasis"

    Article Title: Efficient Propagation of Circulating Tumor Cells: A First Step for Probing Tumor Metastasis

    Journal: Cancers

    doi: 10.3390/cancers12102784

    CTC cultures successfully propagated breast epithelial cells. ( a ) Visual workflow of CTC processing. ( b ) Receptor status of cultured CTCs, based on clinical data. ( c ) Representative phase images (20×) taken after 30 days in culture. Within CTC images, red arrows indicate CTCs and white arrows indicate the adherent leukocyte cells on the top of which CTCs attach and expand. ( d ) RT-PCR was used to confirm that RNA isolated from CTCs was of breast and epithelial origin prior to RNA-seq analysis. ( e ) A box and whisker plot depicting CD45 relative expression following qPCR amplification. CD45 (-) samples were observed to have decreased propagation and did not generate enough high-quality RNA for RNA-sequencing analysis (CTCs 1, 2, 5, 6, 7, and 11).
    Figure Legend Snippet: CTC cultures successfully propagated breast epithelial cells. ( a ) Visual workflow of CTC processing. ( b ) Receptor status of cultured CTCs, based on clinical data. ( c ) Representative phase images (20×) taken after 30 days in culture. Within CTC images, red arrows indicate CTCs and white arrows indicate the adherent leukocyte cells on the top of which CTCs attach and expand. ( d ) RT-PCR was used to confirm that RNA isolated from CTCs was of breast and epithelial origin prior to RNA-seq analysis. ( e ) A box and whisker plot depicting CD45 relative expression following qPCR amplification. CD45 (-) samples were observed to have decreased propagation and did not generate enough high-quality RNA for RNA-sequencing analysis (CTCs 1, 2, 5, 6, 7, and 11).

    Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Isolation, RNA Sequencing Assay, Whisker Assay, Expressing, Real-time Polymerase Chain Reaction, Amplification

    21) Product Images from "Promoter choice: Who should drive the CAR in T cells?"

    Article Title: Promoter choice: Who should drive the CAR in T cells?

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0232915

    The effect of internal promoters in producing functional lentiviral particles. (a) Schematic illustration of the pCCLsin backbone bearing four different internal promoters (CMV, EF-1, hPGK and RPBSA) for driving a long RNA consist of GFP-P2A-Her2CAR-P2A-Mcl-1, (b) HEK293T cells were transfected with lentiviral constructs containing different promoters along with packaging plasmids. At 24 h post transfection, total RNA was extracted and 1 μg of RNA was converted to cDNA. PCR was carried out using specific primers binding to PPT and woodchuck region. Agarose gel electrophoresis displays the PCR product band of each construct. Lower band displays the PCR product of β-actin serving as a loading control. The ratio between viral genomic RNA (vgRNA) to β-actin was quantified and presented in the bar graph (right) using Image Studio Lite. There was no statistically significant difference between promoters (P > 0.05). (c) Shows the ratio between integrated viral cassettes to gDNA 48 h post-transduction. Genomic DNA was extracted from cell lysates and qPCR was performed using Gag for integrated lentivirus and β2 microglobulin and β-actin as a housekeeping genes for host gDNA quantification. There was no statistically significant difference between promoters (P > 0.05). (d) Comparison of the viral particle titration of four different constructs through analysis of the percent GFP expression in HEK293T cells using flow cytometry. Bar graph values represent the titre unit/mL (TU/mL) from three independent repeats. There was no statistically significant difference between promoters (P > 0.05). (e) Transduction efficiency of primary T cells for the four lentivectors. CD3 / CD28 stimulated human primary T cells were transduced at MOI 40 and cells were analyzed for GFP expression at 72 h post-transduction by flow cytometry. A representative experiment, and all GFP MFI values relating to graph, are presented in Fig 3A . Dead cells were excluded with Zombie NIR viability dye gating at analysis. Bar graph values represent the mean values ± SD from three independent repeats. scFv; single-chain variable fragment, VH; variable heavy chain, VL; variable light chain, TM; transmembrane domain.
    Figure Legend Snippet: The effect of internal promoters in producing functional lentiviral particles. (a) Schematic illustration of the pCCLsin backbone bearing four different internal promoters (CMV, EF-1, hPGK and RPBSA) for driving a long RNA consist of GFP-P2A-Her2CAR-P2A-Mcl-1, (b) HEK293T cells were transfected with lentiviral constructs containing different promoters along with packaging plasmids. At 24 h post transfection, total RNA was extracted and 1 μg of RNA was converted to cDNA. PCR was carried out using specific primers binding to PPT and woodchuck region. Agarose gel electrophoresis displays the PCR product band of each construct. Lower band displays the PCR product of β-actin serving as a loading control. The ratio between viral genomic RNA (vgRNA) to β-actin was quantified and presented in the bar graph (right) using Image Studio Lite. There was no statistically significant difference between promoters (P > 0.05). (c) Shows the ratio between integrated viral cassettes to gDNA 48 h post-transduction. Genomic DNA was extracted from cell lysates and qPCR was performed using Gag for integrated lentivirus and β2 microglobulin and β-actin as a housekeeping genes for host gDNA quantification. There was no statistically significant difference between promoters (P > 0.05). (d) Comparison of the viral particle titration of four different constructs through analysis of the percent GFP expression in HEK293T cells using flow cytometry. Bar graph values represent the titre unit/mL (TU/mL) from three independent repeats. There was no statistically significant difference between promoters (P > 0.05). (e) Transduction efficiency of primary T cells for the four lentivectors. CD3 / CD28 stimulated human primary T cells were transduced at MOI 40 and cells were analyzed for GFP expression at 72 h post-transduction by flow cytometry. A representative experiment, and all GFP MFI values relating to graph, are presented in Fig 3A . Dead cells were excluded with Zombie NIR viability dye gating at analysis. Bar graph values represent the mean values ± SD from three independent repeats. scFv; single-chain variable fragment, VH; variable heavy chain, VL; variable light chain, TM; transmembrane domain.

    Techniques Used: Functional Assay, Transfection, Construct, Polymerase Chain Reaction, Binding Assay, Agarose Gel Electrophoresis, Transduction, Real-time Polymerase Chain Reaction, Titration, Expressing, Flow Cytometry

    22) Product Images from "Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation"

    Article Title: Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.03030

    Transcriptional analysis of modABC genes. (A) Expression of β-galactosidase from a P modABC :: lacZ fusion in P. putida J5. Cells were grown aerobically in NI medium supplemented with different concentrations of molybdate. (B) RT-qPCR analysis of the modA gene transcript produced in P. putida J5 that was grown as described above. Results presented in these histograms are the means of three independent experiments, and error bars indicate the standard deviations. Different letters represent significant differences between the treatments.
    Figure Legend Snippet: Transcriptional analysis of modABC genes. (A) Expression of β-galactosidase from a P modABC :: lacZ fusion in P. putida J5. Cells were grown aerobically in NI medium supplemented with different concentrations of molybdate. (B) RT-qPCR analysis of the modA gene transcript produced in P. putida J5 that was grown as described above. Results presented in these histograms are the means of three independent experiments, and error bars indicate the standard deviations. Different letters represent significant differences between the treatments.

    Techniques Used: Expressing, Quantitative RT-PCR, Produced

    23) Product Images from "UDP-Glucose 6-Dehydrogenase Knockout Impairs Migration and Decreases in vivo Metastatic Ability of Breast Cancer Cells"

    Article Title: UDP-Glucose 6-Dehydrogenase Knockout Impairs Migration and Decreases in vivo Metastatic Ability of Breast Cancer Cells

    Journal: bioRxiv

    doi: 10.1101/2020.05.30.125419

    Ugdh-KO does not transcriptionally inhibit EMT in 6DT1 cells. Expression levels of genes associated with EMT in 6DT1 cells quantified by qPCR. Only Cdh1, Fn1 and Six1 are transcriptionally upregulated in Ugdh-KO cells; of these, only Cdh1 upregulation is consistent with decreased EMT. All expression levels are normalized to control gene Tubb5 and displayed relative to WT. Relative quantitation values shown are averages of 4 replicates (2 cell culture replicates × 2 PCR plate replicates). Error bars represent ranges in the relative quantitation values, calculated from standard deviation of corresponding. ΔΔC T values (relative quantitation, RQ = 2^ΔΔC T ). Statistically significant differences between WT and Ugdh-KO are indicated by asterisks: *, p
    Figure Legend Snippet: Ugdh-KO does not transcriptionally inhibit EMT in 6DT1 cells. Expression levels of genes associated with EMT in 6DT1 cells quantified by qPCR. Only Cdh1, Fn1 and Six1 are transcriptionally upregulated in Ugdh-KO cells; of these, only Cdh1 upregulation is consistent with decreased EMT. All expression levels are normalized to control gene Tubb5 and displayed relative to WT. Relative quantitation values shown are averages of 4 replicates (2 cell culture replicates × 2 PCR plate replicates). Error bars represent ranges in the relative quantitation values, calculated from standard deviation of corresponding. ΔΔC T values (relative quantitation, RQ = 2^ΔΔC T ). Statistically significant differences between WT and Ugdh-KO are indicated by asterisks: *, p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, Cell Culture, Polymerase Chain Reaction, Standard Deviation

    24) Product Images from "Deformed Wing Virus spillover from honey bees to bumble bees: a reverse genetic study"

    Article Title: Deformed Wing Virus spillover from honey bees to bumble bees: a reverse genetic study

    Journal: bioRxiv

    doi: 10.1101/2019.12.18.880559

    Morbidity of DWV in bumble bees. A. RT-qPCR analysis of DWV level in developed bumble bees injected with 10 6 GE of DWV at white-eyed pupa stage. Individual values for each sample are shown with dots, lines represent mean ±SD. B. Percentage of visually normal (“ norm” ), discolored (“ disc”) and non-viable (“ nv”) bumble bees developed from pupae in VVD-injected (n=43) and PBS-injected (“Mock”, n=37). C. Phenotype of bumble bees developed from pupae in the incubator.
    Figure Legend Snippet: Morbidity of DWV in bumble bees. A. RT-qPCR analysis of DWV level in developed bumble bees injected with 10 6 GE of DWV at white-eyed pupa stage. Individual values for each sample are shown with dots, lines represent mean ±SD. B. Percentage of visually normal (“ norm” ), discolored (“ disc”) and non-viable (“ nv”) bumble bees developed from pupae in VVD-injected (n=43) and PBS-injected (“Mock”, n=37). C. Phenotype of bumble bees developed from pupae in the incubator.

    Techniques Used: Quantitative RT-PCR, Injection

    Inoculation of bumble bee pupae and larvae with RG-DWV. A. RT-qPCR analysis of DWV accumulation in bumble bee pupae injected with VVV, VDD and VVD DWV. Pupae were injected at white-eyed - “we” - and purple-eyed - “pe” - stages and analyzed 48 h post injection. Each value corresponds to an individual sample analyzed, black lines show mean ±SD. B. Detection of DWV RNA in bumble bee larvae from two different age groups fed with 10 8 GE VVD DWV: qPCR analysis of DWV levels in individual larvae samples, black-circled values correspond to samples which produced a positive result in (-)RNA assay; each value corresponds to an individual sample analyzed, error bars show mean ±SD. DWV (-)RNA assay - strand-specific RT-PCR products run in 1% agarose gel, “+” - positive PCR control for DWV, “M” - molecular weight DNA marker, V - PCR from the DWV inoculum used for larvae feeding.
    Figure Legend Snippet: Inoculation of bumble bee pupae and larvae with RG-DWV. A. RT-qPCR analysis of DWV accumulation in bumble bee pupae injected with VVV, VDD and VVD DWV. Pupae were injected at white-eyed - “we” - and purple-eyed - “pe” - stages and analyzed 48 h post injection. Each value corresponds to an individual sample analyzed, black lines show mean ±SD. B. Detection of DWV RNA in bumble bee larvae from two different age groups fed with 10 8 GE VVD DWV: qPCR analysis of DWV levels in individual larvae samples, black-circled values correspond to samples which produced a positive result in (-)RNA assay; each value corresponds to an individual sample analyzed, error bars show mean ±SD. DWV (-)RNA assay - strand-specific RT-PCR products run in 1% agarose gel, “+” - positive PCR control for DWV, “M” - molecular weight DNA marker, V - PCR from the DWV inoculum used for larvae feeding.

    Techniques Used: Quantitative RT-PCR, Injection, Real-time Polymerase Chain Reaction, Produced, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Molecular Weight, Marker

    25) Product Images from "Consumption of Non-Nutritive Sweetener, Acesulfame Potassium Exacerbates Atherosclerosis through Dysregulation of Lipid Metabolism in ApoE−/− Mice"

    Article Title: Consumption of Non-Nutritive Sweetener, Acesulfame Potassium Exacerbates Atherosclerosis through Dysregulation of Lipid Metabolism in ApoE−/− Mice

    Journal: Nutrients

    doi: 10.3390/nu13113984

    Effects of AceK on proinflammatory cytokine expressions in murine RAW264.7 macrophages. Cells were treated with indicated doses of AceK for 24 h. The cells were harvested and RNA was isolated for the quantification of tumor necrosis factor alpha ( Tnfa ) ( A ), C–C motif chemokine ligand 2 ( Ccl2 ) ( B ), and interleukin-6 ( Il-6 ) ( C ) gene expressions by quantitative PCR ( n = 4).
    Figure Legend Snippet: Effects of AceK on proinflammatory cytokine expressions in murine RAW264.7 macrophages. Cells were treated with indicated doses of AceK for 24 h. The cells were harvested and RNA was isolated for the quantification of tumor necrosis factor alpha ( Tnfa ) ( A ), C–C motif chemokine ligand 2 ( Ccl2 ) ( B ), and interleukin-6 ( Il-6 ) ( C ) gene expressions by quantitative PCR ( n = 4).

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction

    Effects of AceK on lipid metabolism-related gene expressions in HepG2 cells. HepG2 were treated with indicated doses of AceK for 24 h. The cells were then harvested and RNA was isolated for the quantification of acetyl-coA carboxylase ( ACC ) ( A ), fatty acid synthase ( FASN ) ( B ), sterol regulatory element binding protein-1 ( SREBP1 ) ( C ), peroxisomal acyl-coenzyme A oxidase ( ACOX ) ( D ), carnitine palmitoyltransferase-2 ( CPT2 ) ( E ), peroxisome proliferator-activated receptor-α ( PPARA ) ( F ), and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase ( HMGCR ) ( G ) gene expressions by quantitative PCR ( n = 4). ** p
    Figure Legend Snippet: Effects of AceK on lipid metabolism-related gene expressions in HepG2 cells. HepG2 were treated with indicated doses of AceK for 24 h. The cells were then harvested and RNA was isolated for the quantification of acetyl-coA carboxylase ( ACC ) ( A ), fatty acid synthase ( FASN ) ( B ), sterol regulatory element binding protein-1 ( SREBP1 ) ( C ), peroxisomal acyl-coenzyme A oxidase ( ACOX ) ( D ), carnitine palmitoyltransferase-2 ( CPT2 ) ( E ), peroxisome proliferator-activated receptor-α ( PPARA ) ( F ), and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase ( HMGCR ) ( G ) gene expressions by quantitative PCR ( n = 4). ** p

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

    26) Product Images from "Attenuation of canonical NF‐κB signaling maintains function and stability of human Treg"

    Article Title: Attenuation of canonical NF‐κB signaling maintains function and stability of human Treg

    Journal: The Febs Journal

    doi: 10.1111/febs.15361

    Blockade of NF‐κB signaling during Treg induction increases suppressive capacity of iTreg and enhances their stability. (A–C) After 4 days of polarization of co‐tg and IκBmut‐tg T cells with or without Rapa, viable cells were isolated by FACS sorting and (A) cocultured with CPD‐labeled Tresp and activated using anti‐CD3/anti‐CD28 antibodies. After 96 h, proliferation of Tresp was measured via FACS analysis. Left: Statistical analysis was performed with 11 independent donors from a 1 : 1 ratio of Tresp : iTreg. Data are represented as mean ± SD; * P ≤ 0.05 (one‐way ANOVA). Middle: Histogram overlay of one representative donor. Right: Statistical analyses of cocultures of the respective iTreg at the indicated iTreg : Tresp ratios. (B) Genomic DNA of viable cells was isolated, treated with bisulfite for conversion of demethylated cytosines into uracils, and then used in qPCR with specific primers for unconverted (methylated) and converted (demethylated) TSDR of FOXP3, and the demethylation rate of the indicated samples was calculated according to the formula described in the Materials and methods section. Data are presented as heatmap from five individual donors. The heatmap was generated using Heatmapper [ 73 ] ( http://www2.heatmapper.ca/ ). Color code ranges from the lowest measured demethylation rate (4%, blue) to the highest measured demethylation rate (70%, yellow). (C) After 4 days of polarization and an additional 4 days of expansion in only IL‐2 containing medium, cells were FACS‐sorted for viable cells, cocultured with CPD‐labeled Tresp, and activated. After 96 h, proliferation of Tresp was measured via FACS analysis. Left: Statistical analysis was performed with six independent donors. Data are represented as mean ± SD; * P ≤ 0.05 (one‐way ANOVA). Right: Histogram overlay of one representative donor. (D) Percentage of PD‐1 + cells (surface expression) and percentage of FOXP3 + cells (intracellular expression) in IL‐2 expanded iTreg. (E) Percentage of FOXP3 + cells (intracellular expression) following anti‐CD3/anti‐CD28 activation for 24 h. (D, E) Data are represented as mean ± SD; * P ≤ 0.05 (one‐way ANOVA).
    Figure Legend Snippet: Blockade of NF‐κB signaling during Treg induction increases suppressive capacity of iTreg and enhances their stability. (A–C) After 4 days of polarization of co‐tg and IκBmut‐tg T cells with or without Rapa, viable cells were isolated by FACS sorting and (A) cocultured with CPD‐labeled Tresp and activated using anti‐CD3/anti‐CD28 antibodies. After 96 h, proliferation of Tresp was measured via FACS analysis. Left: Statistical analysis was performed with 11 independent donors from a 1 : 1 ratio of Tresp : iTreg. Data are represented as mean ± SD; * P ≤ 0.05 (one‐way ANOVA). Middle: Histogram overlay of one representative donor. Right: Statistical analyses of cocultures of the respective iTreg at the indicated iTreg : Tresp ratios. (B) Genomic DNA of viable cells was isolated, treated with bisulfite for conversion of demethylated cytosines into uracils, and then used in qPCR with specific primers for unconverted (methylated) and converted (demethylated) TSDR of FOXP3, and the demethylation rate of the indicated samples was calculated according to the formula described in the Materials and methods section. Data are presented as heatmap from five individual donors. The heatmap was generated using Heatmapper [ 73 ] ( http://www2.heatmapper.ca/ ). Color code ranges from the lowest measured demethylation rate (4%, blue) to the highest measured demethylation rate (70%, yellow). (C) After 4 days of polarization and an additional 4 days of expansion in only IL‐2 containing medium, cells were FACS‐sorted for viable cells, cocultured with CPD‐labeled Tresp, and activated. After 96 h, proliferation of Tresp was measured via FACS analysis. Left: Statistical analysis was performed with six independent donors. Data are represented as mean ± SD; * P ≤ 0.05 (one‐way ANOVA). Right: Histogram overlay of one representative donor. (D) Percentage of PD‐1 + cells (surface expression) and percentage of FOXP3 + cells (intracellular expression) in IL‐2 expanded iTreg. (E) Percentage of FOXP3 + cells (intracellular expression) following anti‐CD3/anti‐CD28 activation for 24 h. (D, E) Data are represented as mean ± SD; * P ≤ 0.05 (one‐way ANOVA).

    Techniques Used: Isolation, FACS, Labeling, Real-time Polymerase Chain Reaction, Methylation, Generated, Expressing, Activation Assay

    27) Product Images from "Adipose triglyceride lipase–mediated lipid catabolism is essential for bronchiolar regeneration"

    Article Title: Adipose triglyceride lipase–mediated lipid catabolism is essential for bronchiolar regeneration

    Journal: JCI Insight

    doi: 10.1172/jci.insight.149438

    Airways of Atgl -KO/cTg mice show triglyceride accumulation and elevated breathing resistance. ( A ) Representative bronchioles in lung sections from control and Atgl ‑KO/cTg mice stained with ORO. Scatter plots report data on triglyceride (TG) in lung tissues ( n = 3 mice/group), and TGH activity in club cell isolates. The ATGL inhibitor Atglistatin was present if indicated. n = 3 experiments per group. We used 1-way ANOVA with Bonferroni’s test for multiple comparisons. ( B ) In situ Atgl : Images of lung sections incubated with Atgl mRNA–specific probes. Arrowheads depict representative hybridization signals. The scatter plot shows Atgl mRNA expression in LCM bronchioles determined by qPCR and normalized to 18S rRNA. n = 4 mice/group. ( C ) Representative H E sections. Images were computationally modified to illustrate the bronchiolar height measurement process. Scatter plots report data on bronchiolar epithelial height of airways ( n = 9 control mice and n = 8 Atgl-KO/cTg mice). Airway function parameters, resistance and compliance, were measured using a computer-controlled piston ventilator ( n = 6 mice/group). Animals were aged 6 to 9 months. Error bars depict SEM. Statistical analysis was performed with Student’s 2-tailed t test. The outlier (gray) was detected using Grubb’s test (α = 0.05).Scale bars: 20 μm. Detailed information on animals is provided in Supplemental Table 1 .
    Figure Legend Snippet: Airways of Atgl -KO/cTg mice show triglyceride accumulation and elevated breathing resistance. ( A ) Representative bronchioles in lung sections from control and Atgl ‑KO/cTg mice stained with ORO. Scatter plots report data on triglyceride (TG) in lung tissues ( n = 3 mice/group), and TGH activity in club cell isolates. The ATGL inhibitor Atglistatin was present if indicated. n = 3 experiments per group. We used 1-way ANOVA with Bonferroni’s test for multiple comparisons. ( B ) In situ Atgl : Images of lung sections incubated with Atgl mRNA–specific probes. Arrowheads depict representative hybridization signals. The scatter plot shows Atgl mRNA expression in LCM bronchioles determined by qPCR and normalized to 18S rRNA. n = 4 mice/group. ( C ) Representative H E sections. Images were computationally modified to illustrate the bronchiolar height measurement process. Scatter plots report data on bronchiolar epithelial height of airways ( n = 9 control mice and n = 8 Atgl-KO/cTg mice). Airway function parameters, resistance and compliance, were measured using a computer-controlled piston ventilator ( n = 6 mice/group). Animals were aged 6 to 9 months. Error bars depict SEM. Statistical analysis was performed with Student’s 2-tailed t test. The outlier (gray) was detected using Grubb’s test (α = 0.05).Scale bars: 20 μm. Detailed information on animals is provided in Supplemental Table 1 .

    Techniques Used: Mouse Assay, Staining, Activity Assay, In Situ, Incubation, Hybridization, Expressing, Laser Capture Microdissection, Real-time Polymerase Chain Reaction, Modification

    PPARα agonist rescues the mitochondrial club cell phenotype of Atgl -KO/cTg. Mice were pretreated with WY for 2 weeks ( A and D ) or 5 days ( B and C ), if indicated. ( A ) TEM images of club cells. The scatter plots report mitochondrial (M) and lipid droplet (LD) counts. n = 6 mice/group, except Atgl -KO/cTg, n = 7 mice. ( B ) Oxygraph respirometry data of isolated club cells are reported. Mitochondrial complex-substrates were added, as depicted. n = 3 experiments/group. Untreated groups are also shown in Figure 2D . ( C ) Fatty acid β-oxidation of isolated club cells. n = 3 experiments/group. ( D ) PPARα target gene expression in LCM-isolated bronchioles, measured by qPCR, and normalized to 18S rRNA. n = 6 mice/group, except control + WY ( n = 5 mice) and Ucp1 of Atgl ‑KO/cTg ( n = 7 mice). Mice were aged 9 to 11 ( A and D ) or 3 to 5 ( B and C ) months. Error bars depict SEM. Statistical analyses were performed by ordinary 1-way ANOVA with Bonferroni’s test for multiple comparisons, except in B , where Student’s 2-tailed t test was used. The outlier (gray) was detected using Grubb’s test (α = 0.05). Detailed information on animals is provided in Supplemental Table 1 . ADP, adenosine diphosphate; Antimyc., antimycin; Oct. Car., octenoylcarnitine.
    Figure Legend Snippet: PPARα agonist rescues the mitochondrial club cell phenotype of Atgl -KO/cTg. Mice were pretreated with WY for 2 weeks ( A and D ) or 5 days ( B and C ), if indicated. ( A ) TEM images of club cells. The scatter plots report mitochondrial (M) and lipid droplet (LD) counts. n = 6 mice/group, except Atgl -KO/cTg, n = 7 mice. ( B ) Oxygraph respirometry data of isolated club cells are reported. Mitochondrial complex-substrates were added, as depicted. n = 3 experiments/group. Untreated groups are also shown in Figure 2D . ( C ) Fatty acid β-oxidation of isolated club cells. n = 3 experiments/group. ( D ) PPARα target gene expression in LCM-isolated bronchioles, measured by qPCR, and normalized to 18S rRNA. n = 6 mice/group, except control + WY ( n = 5 mice) and Ucp1 of Atgl ‑KO/cTg ( n = 7 mice). Mice were aged 9 to 11 ( A and D ) or 3 to 5 ( B and C ) months. Error bars depict SEM. Statistical analyses were performed by ordinary 1-way ANOVA with Bonferroni’s test for multiple comparisons, except in B , where Student’s 2-tailed t test was used. The outlier (gray) was detected using Grubb’s test (α = 0.05). Detailed information on animals is provided in Supplemental Table 1 . ADP, adenosine diphosphate; Antimyc., antimycin; Oct. Car., octenoylcarnitine.

    Techniques Used: Mouse Assay, Transmission Electron Microscopy, Isolation, Expressing, Laser Capture Microdissection, Real-time Polymerase Chain Reaction

    28) Product Images from "Enhanced vascular activity of a new chimeric promoter containing the full CaMV 35S promoter and the plant XYLOGEN PROTEIN 1 promoter"

    Article Title: Enhanced vascular activity of a new chimeric promoter containing the full CaMV 35S promoter and the plant XYLOGEN PROTEIN 1 promoter

    Journal: 3 Biotech

    doi: 10.1007/s13205-018-1379-8

    Quantitative PCR analysis of β -glucuronidase (GUS) transcriptional expression in vascular and leaf tissues: In lines Z4, uidA is regulated by the promoter of Arabidopsis thaliana XYLOGEN PROTEIN 1; In lines Z5, uidA is regulated by the chimeric promoter 35S-Px. WT, wild-type tobacco; V, vascular tissues of 50- to 60-day-old plants; L, leaves with main vein removed. Experiments were performed in triplicate (biological replicates) and data represent mean relative expression ± standard deviation. Transcription expression between vascular and leaf tissues (paired-samples t -test): For Z4 and Z5lines, *** P = 0.001
    Figure Legend Snippet: Quantitative PCR analysis of β -glucuronidase (GUS) transcriptional expression in vascular and leaf tissues: In lines Z4, uidA is regulated by the promoter of Arabidopsis thaliana XYLOGEN PROTEIN 1; In lines Z5, uidA is regulated by the chimeric promoter 35S-Px. WT, wild-type tobacco; V, vascular tissues of 50- to 60-day-old plants; L, leaves with main vein removed. Experiments were performed in triplicate (biological replicates) and data represent mean relative expression ± standard deviation. Transcription expression between vascular and leaf tissues (paired-samples t -test): For Z4 and Z5lines, *** P = 0.001

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

    Strength of promoters Px and 35S-Px. a Quantitative PCR analysis of β -glucuronidase (GUS) transcriptional expression. Total RNA was extracted from the apical vascular tissues of 50–60-day-old plants. Relative transcription was calculated using the comparative C T ). *** P = 0.000 (Duncan’s test). b Quantitative enzymatic assay of GUS activity in transgenic tobacco lines. The apical vascular tissues of 50–60-day-old plants were also used for soluble protein extraction and measurement of GUS activity. The reaction was carried out at 37 °C for 4 h, and 0.43 mg soluble protein was obtained from each sample. The experiment was repeated at least three times (biological replicates). Bars and whiskers represent mean GUS activity ± standard deviation (SD). *** P = 0.000 (Duncan’s test)
    Figure Legend Snippet: Strength of promoters Px and 35S-Px. a Quantitative PCR analysis of β -glucuronidase (GUS) transcriptional expression. Total RNA was extracted from the apical vascular tissues of 50–60-day-old plants. Relative transcription was calculated using the comparative C T ). *** P = 0.000 (Duncan’s test). b Quantitative enzymatic assay of GUS activity in transgenic tobacco lines. The apical vascular tissues of 50–60-day-old plants were also used for soluble protein extraction and measurement of GUS activity. The reaction was carried out at 37 °C for 4 h, and 0.43 mg soluble protein was obtained from each sample. The experiment was repeated at least three times (biological replicates). Bars and whiskers represent mean GUS activity ± standard deviation (SD). *** P = 0.000 (Duncan’s test)

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Enzymatic Assay, Activity Assay, Transgenic Assay, Protein Extraction, Standard Deviation

    29) Product Images from "The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication"

    Article Title: The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication

    Journal: bioRxiv

    doi: 10.1101/2022.04.14.488435

    Constitutive AKT activity causes defects in viral gene expression and viral DNA synthesis. ( A ) Fibroblasts were induced for expression of either myr-AKT or myr-AKT-K179M (kinase dead) for 24 h and then were infected with HCMV strain TB40/E at MOI = 1. and total RNA were isolated from either myr-AKT or K179M settings at 2, 4, 6, 8, 10, 12, 16, 24, 48, and 72 hpi, including an on-column DNAse I digestion step. RNA samples were reverse-transcribed into cDNA and assayed by qPCR for the abundance of the indicated viral transcripts relative to levels of cellular GAPDH mRNA. ( B ) Total DNA was isolated at the indicated times post infection (hpi) from cells infected exactly as described for panels A and B, and copies of viral DNA were enumerated using quantitative PCR for the HCMV UL69 gene normalized to copies of cellular 18S rDNA loci. ( C ) Infections were set up as in panel A. Cell lysates collected at the indicated times post-infection were assayed by Western blot for the expression of the indicated viral proteins, as well as for the expression of myr-AKT (HA tag) or GAPDH, as a loading control.
    Figure Legend Snippet: Constitutive AKT activity causes defects in viral gene expression and viral DNA synthesis. ( A ) Fibroblasts were induced for expression of either myr-AKT or myr-AKT-K179M (kinase dead) for 24 h and then were infected with HCMV strain TB40/E at MOI = 1. and total RNA were isolated from either myr-AKT or K179M settings at 2, 4, 6, 8, 10, 12, 16, 24, 48, and 72 hpi, including an on-column DNAse I digestion step. RNA samples were reverse-transcribed into cDNA and assayed by qPCR for the abundance of the indicated viral transcripts relative to levels of cellular GAPDH mRNA. ( B ) Total DNA was isolated at the indicated times post infection (hpi) from cells infected exactly as described for panels A and B, and copies of viral DNA were enumerated using quantitative PCR for the HCMV UL69 gene normalized to copies of cellular 18S rDNA loci. ( C ) Infections were set up as in panel A. Cell lysates collected at the indicated times post-infection were assayed by Western blot for the expression of the indicated viral proteins, as well as for the expression of myr-AKT (HA tag) or GAPDH, as a loading control.

    Techniques Used: Activity Assay, Expressing, DNA Synthesis, Infection, Isolation, Real-time Polymerase Chain Reaction, Western Blot

    30) Product Images from "Exosomal mitochondrial tRNAs and miRNAs as potential predictors of inflammation in renal proximal tubular epithelial cells"

    Article Title: Exosomal mitochondrial tRNAs and miRNAs as potential predictors of inflammation in renal proximal tubular epithelial cells

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2022.04.035

    Regulation of candidate exosomal miRNAs and mt-tRNAs from RPTECs by combinatorial treatment of GW4869 and manumycin A (A–C) RT-qPCR analysis of dysregulated exosomal miRNAs, hsa-miR-21-5p, -215-5p, and -192-5p, derived from non-stimulated (CK−) or cytokine-stimulated (CK+) RPTECs treated with both inhibitors (2× inhib.), GW4869 (5 μM), and manumycin A (125 nM). The percentage release of each candidate exosomal miRNA was calculated by obtaining the total RNA copies in exosomes and normalized to the total RNA copies in cells (set to 100%) for each treatment (see section “ materials and methods ”). Data represent mean ± SD, which consists of three independent samples pooled together. (D–F) The percentage release of each candidate exosomal mt-tRNAs, mt-tRNA His , mt-tRNA Leu1 , and mt-tRNA Val was analyzed similar to (A–C). Data represent mean ± SEM of two independent experiments.
    Figure Legend Snippet: Regulation of candidate exosomal miRNAs and mt-tRNAs from RPTECs by combinatorial treatment of GW4869 and manumycin A (A–C) RT-qPCR analysis of dysregulated exosomal miRNAs, hsa-miR-21-5p, -215-5p, and -192-5p, derived from non-stimulated (CK−) or cytokine-stimulated (CK+) RPTECs treated with both inhibitors (2× inhib.), GW4869 (5 μM), and manumycin A (125 nM). The percentage release of each candidate exosomal miRNA was calculated by obtaining the total RNA copies in exosomes and normalized to the total RNA copies in cells (set to 100%) for each treatment (see section “ materials and methods ”). Data represent mean ± SD, which consists of three independent samples pooled together. (D–F) The percentage release of each candidate exosomal mt-tRNAs, mt-tRNA His , mt-tRNA Leu1 , and mt-tRNA Val was analyzed similar to (A–C). Data represent mean ± SEM of two independent experiments.

    Techniques Used: Quantitative RT-PCR, Derivative Assay, Inhibition

    Validation of dysregulated exosomal mt-tRNAs, mt-tRNA His , mt-tRNA Leu2 , mt-tRNA Val , mt-tRNA Phe , and mt-tRNA Leu1 derived from an RPTEC model system of renal inflammation (A) RT-qPCR analysis of total exosomal RNA extracts from either non-stimulated (CK−) or cytokine-stimulated (CK+) RPTECs (see section “ materials and methods ”). The FC expression of each candidate, relative to CK− samples, was analyzed by the 2 −ΔΔCt method. Data indicate mean ± SEM of four independent experiments. Statistical analysis of ΔCt values was performed using two-way ANOVA with multiple comparison test and Bonferroni correction (GraphPad Prism 8.0.1). ∗∗∗∗p
    Figure Legend Snippet: Validation of dysregulated exosomal mt-tRNAs, mt-tRNA His , mt-tRNA Leu2 , mt-tRNA Val , mt-tRNA Phe , and mt-tRNA Leu1 derived from an RPTEC model system of renal inflammation (A) RT-qPCR analysis of total exosomal RNA extracts from either non-stimulated (CK−) or cytokine-stimulated (CK+) RPTECs (see section “ materials and methods ”). The FC expression of each candidate, relative to CK− samples, was analyzed by the 2 −ΔΔCt method. Data indicate mean ± SEM of four independent experiments. Statistical analysis of ΔCt values was performed using two-way ANOVA with multiple comparison test and Bonferroni correction (GraphPad Prism 8.0.1). ∗∗∗∗p

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

    31) Product Images from "Targeting Nup358/RanBP2 by a viral protein disrupts stress granule formation"

    Article Title: Targeting Nup358/RanBP2 by a viral protein disrupts stress granule formation

    Journal: bioRxiv

    doi: 10.1101/2022.05.19.492599

    Nup358 promotes CrPV infection in an R146-dependent manner. (A) Immunoblots of S2 cells treated with dsRNA targeting GP210, mTor, Rae1, Nup88, Nup214, Nup358 or control FLuc, followed by mock infection or infection with wild-type or mutant CrPV virus for 8 hours (MOI 1). (B) Quantification of VP2 intensity normalized to tubulin. The intensity values are normalized to the VP2/Tubulin intensity in FLuc control knockdown cells. (C) CrPV viral RNA levels by qRT-PCR analysis normalized to Rps9 mRNA levels. Data are mean ± SD relative to WT p > 0.05 (ns), p
    Figure Legend Snippet: Nup358 promotes CrPV infection in an R146-dependent manner. (A) Immunoblots of S2 cells treated with dsRNA targeting GP210, mTor, Rae1, Nup88, Nup214, Nup358 or control FLuc, followed by mock infection or infection with wild-type or mutant CrPV virus for 8 hours (MOI 1). (B) Quantification of VP2 intensity normalized to tubulin. The intensity values are normalized to the VP2/Tubulin intensity in FLuc control knockdown cells. (C) CrPV viral RNA levels by qRT-PCR analysis normalized to Rps9 mRNA levels. Data are mean ± SD relative to WT p > 0.05 (ns), p

    Techniques Used: Infection, Western Blot, Mutagenesis, Quantitative RT-PCR

    32) Product Images from "Evidence for and against deformed wing virus spillover from honey bees to bumble bees: a reverse genetic analysis"

    Article Title: Evidence for and against deformed wing virus spillover from honey bees to bumble bees: a reverse genetic analysis

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-73809-3

    Morbidity of DWV in bumble bees. ( a ) qPCR analysis of DWV level in developed bumble bees injected with 10 6 GE of VVD DWV at white-eyed (P0-P1) pupa stage. Individual values for each sample are shown with dots, lines represent mean ± SD, “GE” —genome equivalents. ( b ) Percentage of visually normal (“ norm” ), discolored (“ disc”) and non-viable (“ nv”) bumble bees developed from pupae in VVD DWV-injected (n = 43) and PBS-injected (“Mock”, n = 37) groups. ( c ) Phenotype of bumble bees developed from pupae in the incubator.
    Figure Legend Snippet: Morbidity of DWV in bumble bees. ( a ) qPCR analysis of DWV level in developed bumble bees injected with 10 6 GE of VVD DWV at white-eyed (P0-P1) pupa stage. Individual values for each sample are shown with dots, lines represent mean ± SD, “GE” —genome equivalents. ( b ) Percentage of visually normal (“ norm” ), discolored (“ disc”) and non-viable (“ nv”) bumble bees developed from pupae in VVD DWV-injected (n = 43) and PBS-injected (“Mock”, n = 37) groups. ( c ) Phenotype of bumble bees developed from pupae in the incubator.

    Techniques Used: Real-time Polymerase Chain Reaction, Injection

    Inoculation of bumble bee pupae and larvae with RG-DWV. ( a ) qPCR analysis of DWV accumulation in bumble bee pupae injected with VVV, VDD and VVD DWV. Pupae were injected at white-eyed (P0-P1)—“we”—and brown-eyed (P7-P8)—“be”—stages and analyzed 48 h post injection. Each value corresponds to an individual sample analyzed, black lines show mean ± SD, “ GE ” —genome equivalents. ( b ) Detection of DWV RNA in bumble bee larvae from two different age groups fed with 10 8 GE VVD DWV: qPCR analysis of DWV levels in individual larvae samples, black-circled values correspond to samples which produced a positive result in (−)RNA assay; each value corresponds to an individual sample analyzed, error bars show mean ± SD, “ GE ” —genome equivalents. DWV (-)RNA assay—PCR products obtained after strand-specific reverse transcription and run in 1% agarose gel, “ + ”—positive PCR control for DWV, “M”—molecular weight DNA marker, “V”—PCR from the DWV inoculum used for larvae feeding.
    Figure Legend Snippet: Inoculation of bumble bee pupae and larvae with RG-DWV. ( a ) qPCR analysis of DWV accumulation in bumble bee pupae injected with VVV, VDD and VVD DWV. Pupae were injected at white-eyed (P0-P1)—“we”—and brown-eyed (P7-P8)—“be”—stages and analyzed 48 h post injection. Each value corresponds to an individual sample analyzed, black lines show mean ± SD, “ GE ” —genome equivalents. ( b ) Detection of DWV RNA in bumble bee larvae from two different age groups fed with 10 8 GE VVD DWV: qPCR analysis of DWV levels in individual larvae samples, black-circled values correspond to samples which produced a positive result in (−)RNA assay; each value corresponds to an individual sample analyzed, error bars show mean ± SD, “ GE ” —genome equivalents. DWV (-)RNA assay—PCR products obtained after strand-specific reverse transcription and run in 1% agarose gel, “ + ”—positive PCR control for DWV, “M”—molecular weight DNA marker, “V”—PCR from the DWV inoculum used for larvae feeding.

    Techniques Used: Real-time Polymerase Chain Reaction, Injection, Produced, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Marker

    33) Product Images from "Neuroligin-mediated neurodevelopmental defects are induced by mitochondrial dysfunction and prevented by lutein in C. elegans"

    Article Title: Neuroligin-mediated neurodevelopmental defects are induced by mitochondrial dysfunction and prevented by lutein in C. elegans

    Journal: Nature Communications

    doi: 10.1038/s41467-022-29972-4

    Upregulation of neuroligin and neurexin genes upon nuo-5 is conserved across species and the defect is rescued by lutein but no other antioxidants. a Expression levels of neuroligin (NLGN1-2 and 4) and neurexin (NRX1 and 3) transcripts in brains of NDUFS4−/− mice compared to healthy animals normalized to ACTB, assessed by qPCR. Three biologically independent experiments, n = 3. NLGN1 p = 0.0245, NLGN2 p = 0.019, NLGN4.1 p = 0.0179, NLGN4.2 p = 0.0104, NRXN1 p = 0.0013, NRXN3 p = 0.0068. b , c Fold change of genes described as nlg-1 interactors (from microarray data). d Quantification of GFP expression of p nlg-1 transgenic strain. Box plots indicate median (middle line), 25th, 75th percentile (box) and 5th and 95th percentile (whiskers) as well as outliers (single points). n ≥ 47 over three biologically independent experiments. con vs nuo-5 p
    Figure Legend Snippet: Upregulation of neuroligin and neurexin genes upon nuo-5 is conserved across species and the defect is rescued by lutein but no other antioxidants. a Expression levels of neuroligin (NLGN1-2 and 4) and neurexin (NRX1 and 3) transcripts in brains of NDUFS4−/− mice compared to healthy animals normalized to ACTB, assessed by qPCR. Three biologically independent experiments, n = 3. NLGN1 p = 0.0245, NLGN2 p = 0.019, NLGN4.1 p = 0.0179, NLGN4.2 p = 0.0104, NRXN1 p = 0.0013, NRXN3 p = 0.0068. b , c Fold change of genes described as nlg-1 interactors (from microarray data). d Quantification of GFP expression of p nlg-1 transgenic strain. Box plots indicate median (middle line), 25th, 75th percentile (box) and 5th and 95th percentile (whiskers) as well as outliers (single points). n ≥ 47 over three biologically independent experiments. con vs nuo-5 p

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Microarray, Transgenic Assay

    34) Product Images from "TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism"

    Article Title: TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism

    Journal: Open Biology

    doi: 10.1098/rsob.170134

    Timed induction of bmp4 affects expression of BMP antagonists. ( a ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of tg(hsp70:bmp4, cmlc2:GFP) embryos at 30 hpf after heat shock at 24 hpf as represented by the log2(fold change) of individual samples. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 0.170 and 0.049, respectively. ( b , c ) WMISH for fsta (30 hpf) in tg(hsp70:bmp4, cmlc2:GFP) after heat shock at 24 hpf ( b ) and control embryos ( c ). Strong upregulation after the heat shock is seen in the optic fissure and other expression domains, but not the ciliary marginal zone (arrow). WMISHs in ( b ) and ( c ) were stained in parallel for the same amount of time.
    Figure Legend Snippet: Timed induction of bmp4 affects expression of BMP antagonists. ( a ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of tg(hsp70:bmp4, cmlc2:GFP) embryos at 30 hpf after heat shock at 24 hpf as represented by the log2(fold change) of individual samples. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 0.170 and 0.049, respectively. ( b , c ) WMISH for fsta (30 hpf) in tg(hsp70:bmp4, cmlc2:GFP) after heat shock at 24 hpf ( b ) and control embryos ( c ). Strong upregulation after the heat shock is seen in the optic fissure and other expression domains, but not the ciliary marginal zone (arrow). WMISHs in ( b ) and ( c ) were stained in parallel for the same amount of time.

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

    BMP antagonists grem2b and fsta are expressed in the optic fissure. WMISHs were performed at 30 hpf and are shown in lateral view. ( a ) bmp4 is expressed in the dorsal optic cup (arrow). ( b ) Expression of vax2 in the ventral retina. ( c , d ) Expression of grem2b in the optic cup is restricted to the optic fissure (arrow). ( d ) Confocal view of grem2b expression (red) with DAPI counterstaining (blue). ( e , f ) fsta is expressed in the optic fissure (arrows), as well as the ciliary marginal zone (CMZ, arrowhead). ( f ) Confocal view of fsta expression (red) with DAPI counterstaining (blue). ( g ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of SIS3-treated embryos (30 hpf) as represented by the log2(fold change) of individual samples. Embryos were treated from 24 hpf onward, controls were treated with DMSO. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 4.3 × 10 −3 and 0.877, respectively. ( h ) Model of the proposed role of TGFβ and BMP antagonism during optic fissure fusion. TGFβ signalling domains in the optic fissure margins are shielded from BMP by induced BMP antagonists. ( i ) Scheme of a heat shock inducible BMP construct used to create the transgenic line tg(hsp70:bmp4, cmlc2:GFP) . GFP expressed under the cardiac cmlc2 promoter serves as transgenesis marker. Experimental procedure using heat shocks at different time points between 21 and 26 hpf to induce bmp4 expression, aiming at disrupting optic fissure fusion. Analysis of phenotypes was scheduled for 3 dpf.
    Figure Legend Snippet: BMP antagonists grem2b and fsta are expressed in the optic fissure. WMISHs were performed at 30 hpf and are shown in lateral view. ( a ) bmp4 is expressed in the dorsal optic cup (arrow). ( b ) Expression of vax2 in the ventral retina. ( c , d ) Expression of grem2b in the optic cup is restricted to the optic fissure (arrow). ( d ) Confocal view of grem2b expression (red) with DAPI counterstaining (blue). ( e , f ) fsta is expressed in the optic fissure (arrows), as well as the ciliary marginal zone (CMZ, arrowhead). ( f ) Confocal view of fsta expression (red) with DAPI counterstaining (blue). ( g ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of SIS3-treated embryos (30 hpf) as represented by the log2(fold change) of individual samples. Embryos were treated from 24 hpf onward, controls were treated with DMSO. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 4.3 × 10 −3 and 0.877, respectively. ( h ) Model of the proposed role of TGFβ and BMP antagonism during optic fissure fusion. TGFβ signalling domains in the optic fissure margins are shielded from BMP by induced BMP antagonists. ( i ) Scheme of a heat shock inducible BMP construct used to create the transgenic line tg(hsp70:bmp4, cmlc2:GFP) . GFP expressed under the cardiac cmlc2 promoter serves as transgenesis marker. Experimental procedure using heat shocks at different time points between 21 and 26 hpf to induce bmp4 expression, aiming at disrupting optic fissure fusion. Analysis of phenotypes was scheduled for 3 dpf.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Construct, Transgenic Assay, Marker

    Loss of TGFβ2 ligand results in coloboma. ( a , b ) Frontal sections (E18.5, H E) of ( b ) TGFβ2 KO and ( a ) wild-type mouse embryos from mixed genetic background. Note the persisting optic fissure (boxed in b ), scale bars 200 µm. ( c , d ) Frontal sections (E16.5, H E) of ( d ) TGFβ2 KO and ( c ) wild-type mouse embryos from a sole genetic background, mild coloboma phenotype boxed in ( d ), Scale bars 200 µm. ( e ) Expression analysis of gremlin and follistatin, decrease in TGFβ2 KO (TGFβ2/GDNF KO) as represented by the log2(fold change). Error bars represent the 95% confidence interval. Corrected p -values of control gene expression compared to KO for Grem1 and Fst, 5.5 × 10 −3 and 1.2 × 10 −3 , respectively. ( f ) Expression analysis of gremlin and follistatin by quantitative PCR, decrease in TGFβ2 KO (TGFβ2 −/−, GDNF +/− ) compared to controls (TGFβ2 +/+ , GDNF +/− ) as represented by the log2(fold change) of individual samples; n = 3, horizontal bars represent the arithmetic mean. p -values for Grem1 and Fst, 7.8 × 10 −3 and 0.029, respectively. ( g ) Selected terms enriched in the set of downregulated genes in TGFβ2/GDNF KO microarray based on gProfiler analysis. BP, biological process; CC, cellular component; MF, molecular function. ( h ) Expression analysis of several ECM-related genes. Error bars represent the 95% confidence interval. ( i ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in TGFβ2 KO (sole genetic background) compared to wild-type as represented by the log2(fold change) of individual samples; n = 5, one KO sample was excluded as an outlier. Horizontal bars represent the arithmetic mean. p -Values for Grem1 and Fst, 0.312 and 0.027, respectively.
    Figure Legend Snippet: Loss of TGFβ2 ligand results in coloboma. ( a , b ) Frontal sections (E18.5, H E) of ( b ) TGFβ2 KO and ( a ) wild-type mouse embryos from mixed genetic background. Note the persisting optic fissure (boxed in b ), scale bars 200 µm. ( c , d ) Frontal sections (E16.5, H E) of ( d ) TGFβ2 KO and ( c ) wild-type mouse embryos from a sole genetic background, mild coloboma phenotype boxed in ( d ), Scale bars 200 µm. ( e ) Expression analysis of gremlin and follistatin, decrease in TGFβ2 KO (TGFβ2/GDNF KO) as represented by the log2(fold change). Error bars represent the 95% confidence interval. Corrected p -values of control gene expression compared to KO for Grem1 and Fst, 5.5 × 10 −3 and 1.2 × 10 −3 , respectively. ( f ) Expression analysis of gremlin and follistatin by quantitative PCR, decrease in TGFβ2 KO (TGFβ2 −/−, GDNF +/− ) compared to controls (TGFβ2 +/+ , GDNF +/− ) as represented by the log2(fold change) of individual samples; n = 3, horizontal bars represent the arithmetic mean. p -values for Grem1 and Fst, 7.8 × 10 −3 and 0.029, respectively. ( g ) Selected terms enriched in the set of downregulated genes in TGFβ2/GDNF KO microarray based on gProfiler analysis. BP, biological process; CC, cellular component; MF, molecular function. ( h ) Expression analysis of several ECM-related genes. Error bars represent the 95% confidence interval. ( i ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in TGFβ2 KO (sole genetic background) compared to wild-type as represented by the log2(fold change) of individual samples; n = 5, one KO sample was excluded as an outlier. Horizontal bars represent the arithmetic mean. p -Values for Grem1 and Fst, 0.312 and 0.027, respectively.

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

    35) Product Images from "Promoter Choice: Who Should Drive the CAR in T Cells?"

    Article Title: Promoter Choice: Who Should Drive the CAR in T Cells?

    Journal: bioRxiv

    doi: 10.1101/2020.04.27.063743

    The effect of internal promoters in producing functional lentiviral particles a) Schematic illustration of the pCCLsin backbone bearing four different internal promoters (CMV, EF-1, hPGK and RPBSA) for driving a long RNA consist of GFP-P2A-Her2CAR-P2A-Mcl-1 b) HEK293T cells were transfected with lentiviral constructs containing different promoters along with packaging plasmids. At 24 h post transfection, total RNA was extracted and 1 μg of RNA was converted to cDNA. PCR was carried out using specific primers binding to PPT and woodchuck region. Agarose gel electrophoresis displays the PCR product band of each construct. Lower band displays the PCR product of β-actin serving as a loading control. c) Ratio between integrated viral cassettes to gDNA 48 h post-transduction. Genomic DNA was extracted from cell lysates and qPCR was performed using Gag for integrated lentivirus, β2 microglobulin for gDNA and β-actin as a housekeeping gene. d) Comparing the viral titration of four different constructs by analyzing the percentage of GFP expression in HEK293T cells using flow cytometry. Bar graph values represent the titre unit/mL (TU/mL) from three independent repeats e) Transduction efficacy of primary T cells for four lentivectors. CD3/ CD28 stimulated human primary T cells were transduced at MOI 40 and cells were analyzed for GFP expression 72 h after transduction by flow cytometry. Dead cells were excluded with Zombie NIR viability dye gating at analysis. Bar graph values represent the mean values ± SD from three different repeats.
    Figure Legend Snippet: The effect of internal promoters in producing functional lentiviral particles a) Schematic illustration of the pCCLsin backbone bearing four different internal promoters (CMV, EF-1, hPGK and RPBSA) for driving a long RNA consist of GFP-P2A-Her2CAR-P2A-Mcl-1 b) HEK293T cells were transfected with lentiviral constructs containing different promoters along with packaging plasmids. At 24 h post transfection, total RNA was extracted and 1 μg of RNA was converted to cDNA. PCR was carried out using specific primers binding to PPT and woodchuck region. Agarose gel electrophoresis displays the PCR product band of each construct. Lower band displays the PCR product of β-actin serving as a loading control. c) Ratio between integrated viral cassettes to gDNA 48 h post-transduction. Genomic DNA was extracted from cell lysates and qPCR was performed using Gag for integrated lentivirus, β2 microglobulin for gDNA and β-actin as a housekeeping gene. d) Comparing the viral titration of four different constructs by analyzing the percentage of GFP expression in HEK293T cells using flow cytometry. Bar graph values represent the titre unit/mL (TU/mL) from three independent repeats e) Transduction efficacy of primary T cells for four lentivectors. CD3/ CD28 stimulated human primary T cells were transduced at MOI 40 and cells were analyzed for GFP expression 72 h after transduction by flow cytometry. Dead cells were excluded with Zombie NIR viability dye gating at analysis. Bar graph values represent the mean values ± SD from three different repeats.

    Techniques Used: Functional Assay, Transfection, Construct, Polymerase Chain Reaction, Binding Assay, Agarose Gel Electrophoresis, Transduction, Real-time Polymerase Chain Reaction, Titration, Expressing, Flow Cytometry

    36) Product Images from "Silencing cryptic specialized metabolism in Streptomyces by the nucleoid-associated protein Lsr2"

    Article Title: Silencing cryptic specialized metabolism in Streptomyces by the nucleoid-associated protein Lsr2

    Journal: eLife

    doi: 10.7554/eLife.47691

    Dominant negative Lsr2 variant (Lsr2*) inhibits DNA binding by wild type Lsr2 and promotes antibiotic production in S. venezuelae . ( A ) Electrophoretic mobility shift assay illustrating DNA binding by wild type Lsr2, no DNA binding by the R82A mutant allele (mutant Lsr2*), and effective inhibition of Lsr2 binding by the mutant variant, based on the increasing abundance of ‘free probe’ as mutant concentrations rise. Probe: 1.5 kb region corresponding to the Lsr2 binding site in the chloramphenicol biosynthetic cluster. Reactions were separated on a 1% agarose gel stained with ethidium bromide. ( B ) Bioactivity of S. venezuelae extracts against Micrococcus luteus . Wild type S. venezuelae carrying either a control (empty) plasmid (left), or one overexpressing the mutant 9R82A) Lsr2 variant (right; Lsr2*) were cultured for 18 hr prior to extraction in methanol and reconstitution in DMSO. Extracts were applied to Whatman filter discs, and cultures were grown overnight. ( C ) Quantitative PCR analysis of Lsr2-3xFLAG-binding to the validated target site sven_0926 , following ChIP, for a strain carrying an empty plasmid (control) or overexpressing Lsr2* (O/E Lsr2*). Overexpressing Lsr2* led to a 40% drop in binding by Lsr2. ChIP experiments were conducted in duplicate, and qPCR was done for each replicate in technical triplicate.
    Figure Legend Snippet: Dominant negative Lsr2 variant (Lsr2*) inhibits DNA binding by wild type Lsr2 and promotes antibiotic production in S. venezuelae . ( A ) Electrophoretic mobility shift assay illustrating DNA binding by wild type Lsr2, no DNA binding by the R82A mutant allele (mutant Lsr2*), and effective inhibition of Lsr2 binding by the mutant variant, based on the increasing abundance of ‘free probe’ as mutant concentrations rise. Probe: 1.5 kb region corresponding to the Lsr2 binding site in the chloramphenicol biosynthetic cluster. Reactions were separated on a 1% agarose gel stained with ethidium bromide. ( B ) Bioactivity of S. venezuelae extracts against Micrococcus luteus . Wild type S. venezuelae carrying either a control (empty) plasmid (left), or one overexpressing the mutant 9R82A) Lsr2 variant (right; Lsr2*) were cultured for 18 hr prior to extraction in methanol and reconstitution in DMSO. Extracts were applied to Whatman filter discs, and cultures were grown overnight. ( C ) Quantitative PCR analysis of Lsr2-3xFLAG-binding to the validated target site sven_0926 , following ChIP, for a strain carrying an empty plasmid (control) or overexpressing Lsr2* (O/E Lsr2*). Overexpressing Lsr2* led to a 40% drop in binding by Lsr2. ChIP experiments were conducted in duplicate, and qPCR was done for each replicate in technical triplicate.

    Techniques Used: Dominant Negative Mutation, Variant Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Mutagenesis, Inhibition, Agarose Gel Electrophoresis, Staining, Plasmid Preparation, Cell Culture, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    37) Product Images from "Interferon-α subtype treatment induces the repression of SRSF1 in HIV-1 target cells and affects HIV-1 post integration steps"

    Article Title: Interferon-α subtype treatment induces the repression of SRSF1 in HIV-1 target cells and affects HIV-1 post integration steps

    Journal: bioRxiv

    doi: 10.1101/2021.06.11.448031

    siRNA-induced knockdown of SRSF1. HEK293T cells were transfected with the proviral clone pNL4-3 PI952 ( 54 ) and the indicated siRNA. 72 h post transfection, cells were harvested and RNA and viral supernatant isolated. a) – b) Isolated RNA was subjected to RT-qPCR. Relative mRNA expression levels of a) SRSF1 and b) Exon 1 and Exon 7 containing mRNAs (total viral mRNA) normalized to GAPDH. c) Isolated RNA was subjected to RT-PCR using the indicated primer pairs for the 2 kb-, 4 kb- and tat mRNA-class. HIV-1 transcript isoforms are depicted on the right. To compare total RNA amounts, separate RT-PCRs amplifying HIV-1 exon 7 containing transcripts as well as cellular GAPDH were performed. PCR amplicons were separated on a 12% nondenaturing polyacrylamide gel and stained with Midori green Advance DNA stain (Nippon Genetics). d) – g) RT-qPCR results for relative mRNA expression levels of d) vif and vpr, e) tat1, tat2 and tat3, f) Exon 2 and Exon 3 containing and g) multiply spliced and unspliced mRNAs. HIV-1 mRNAs were analyzed using the indicated primers ( Table 1 ). The splicing pattern of pNL4-3 PI952 was set to 100% and the relative splice site usage was normalized to total viral mRNA levels (Exon 7). Unpaired t tests were calculated to determine whether the difference between the group of samples reached the level of statistical significance (* p
    Figure Legend Snippet: siRNA-induced knockdown of SRSF1. HEK293T cells were transfected with the proviral clone pNL4-3 PI952 ( 54 ) and the indicated siRNA. 72 h post transfection, cells were harvested and RNA and viral supernatant isolated. a) – b) Isolated RNA was subjected to RT-qPCR. Relative mRNA expression levels of a) SRSF1 and b) Exon 1 and Exon 7 containing mRNAs (total viral mRNA) normalized to GAPDH. c) Isolated RNA was subjected to RT-PCR using the indicated primer pairs for the 2 kb-, 4 kb- and tat mRNA-class. HIV-1 transcript isoforms are depicted on the right. To compare total RNA amounts, separate RT-PCRs amplifying HIV-1 exon 7 containing transcripts as well as cellular GAPDH were performed. PCR amplicons were separated on a 12% nondenaturing polyacrylamide gel and stained with Midori green Advance DNA stain (Nippon Genetics). d) – g) RT-qPCR results for relative mRNA expression levels of d) vif and vpr, e) tat1, tat2 and tat3, f) Exon 2 and Exon 3 containing and g) multiply spliced and unspliced mRNAs. HIV-1 mRNAs were analyzed using the indicated primers ( Table 1 ). The splicing pattern of pNL4-3 PI952 was set to 100% and the relative splice site usage was normalized to total viral mRNA levels (Exon 7). Unpaired t tests were calculated to determine whether the difference between the group of samples reached the level of statistical significance (* p

    Techniques Used: Transfection, Isolation, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Staining

    HIV-1 NL4-3 genome. a) HIV-1 genome with open reading frames (ORFs) and long terminal repeats (LTRs). 5’- and 3’-splice sites are indicated as well as the Rev response element (RRE). Vif Exon and ORF is highlighted in red. b) Vif and Vpr mRNs are spliced from 5’-ss D1 to 3’-ss A1 and 5’-ss D1 to 3’-ss A2 respectively, harboring the non-coding leader Exons 2 and 3. AUG-containing Introns 2 and 3 are contained respectively. c) Binding sites of primers for RT-qPCR and RT-PCR. Grey boxes indicate Exons, while straight lines indicate Introns. Black arrowheads indicate primers. Primers with black rectangle and black arrowhead connected via dashed line indicate Exon-junction primers.
    Figure Legend Snippet: HIV-1 NL4-3 genome. a) HIV-1 genome with open reading frames (ORFs) and long terminal repeats (LTRs). 5’- and 3’-splice sites are indicated as well as the Rev response element (RRE). Vif Exon and ORF is highlighted in red. b) Vif and Vpr mRNs are spliced from 5’-ss D1 to 3’-ss A1 and 5’-ss D1 to 3’-ss A2 respectively, harboring the non-coding leader Exons 2 and 3. AUG-containing Introns 2 and 3 are contained respectively. c) Binding sites of primers for RT-qPCR and RT-PCR. Grey boxes indicate Exons, while straight lines indicate Introns. Black arrowheads indicate primers. Primers with black rectangle and black arrowhead connected via dashed line indicate Exon-junction primers.

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

    Overexpression of SRSF1. HEK293T cells were transfected with the proviral clone pNL4-3 PI952 ( 54 ) and pcDNA-FLAG-SF2 ( 65 ). 72 h post transfection, cells were harvested and RNA and viral supernatant isolated. a) – b) Isolated RNA was subjected to RT-qPCR. Relative mRNA expression levels of a) SRSF1 and b) Exon 1 and Exon 7 containing mRNAs (total viral mRNA) normalized to GAPDH. c) Vero cells were transiently co-transfected with pTA-Luc-NL4-3, pSVctat ( 66 ) and pEGFP-SF2 ( 67 ) at the indicated concentrations. Activity of HIV-1 LTR promoter was measured via luminescent read-out. d) Isolated RNA was subjected to RT-PCR using the indicated primer pairs for the 2 kb-, 4 kb- and tat mRNA-class. HIV-1 transcript isoforms are depicted on the right. To compare total RNA amounts, separate RT-PCRs amplifying HIV-1 exon 7 containing transcripts as well as cellular GAPDH were performed. PCR amplicons were separated on a 12% nondenaturing polyacrylamide gel and stained with Midori green Advance DNA stain (Nippon Genetics). e) – h) RT-qPCR results for relative mRNA expression levels of e) vif and vpr, f) tat1, tat2 and tat3, g) Exon 2 and Exon 3 containing and h) multiply spliced and unspliced mRNAs. HIV-1 mRNAs were analyzed using the indicated primers ( Table 1 ). The splicing pattern of pNL4-3 PI952 was set to 100% and the relative splice site usage was normalized to total viral mRNA levels (Exon 7). Unpaired t tests were calculated to determine whether the difference between the group of samples reached the level of statistical significance (* p
    Figure Legend Snippet: Overexpression of SRSF1. HEK293T cells were transfected with the proviral clone pNL4-3 PI952 ( 54 ) and pcDNA-FLAG-SF2 ( 65 ). 72 h post transfection, cells were harvested and RNA and viral supernatant isolated. a) – b) Isolated RNA was subjected to RT-qPCR. Relative mRNA expression levels of a) SRSF1 and b) Exon 1 and Exon 7 containing mRNAs (total viral mRNA) normalized to GAPDH. c) Vero cells were transiently co-transfected with pTA-Luc-NL4-3, pSVctat ( 66 ) and pEGFP-SF2 ( 67 ) at the indicated concentrations. Activity of HIV-1 LTR promoter was measured via luminescent read-out. d) Isolated RNA was subjected to RT-PCR using the indicated primer pairs for the 2 kb-, 4 kb- and tat mRNA-class. HIV-1 transcript isoforms are depicted on the right. To compare total RNA amounts, separate RT-PCRs amplifying HIV-1 exon 7 containing transcripts as well as cellular GAPDH were performed. PCR amplicons were separated on a 12% nondenaturing polyacrylamide gel and stained with Midori green Advance DNA stain (Nippon Genetics). e) – h) RT-qPCR results for relative mRNA expression levels of e) vif and vpr, f) tat1, tat2 and tat3, g) Exon 2 and Exon 3 containing and h) multiply spliced and unspliced mRNAs. HIV-1 mRNAs were analyzed using the indicated primers ( Table 1 ). The splicing pattern of pNL4-3 PI952 was set to 100% and the relative splice site usage was normalized to total viral mRNA levels (Exon 7). Unpaired t tests were calculated to determine whether the difference between the group of samples reached the level of statistical significance (* p

    Techniques Used: Over Expression, Transfection, Isolation, Quantitative RT-PCR, Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Staining

    38) Product Images from "CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding"

    Article Title: CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding

    Journal: Communications Biology

    doi: 10.1038/s42003-019-0288-7

    Evaluation of genome-edited and wild type non-edited control plants of Gonja Manjaya for induction of BSV symptoms under water stress conditions. Two-month-old plants were subjected to water stress for 2 weeks. Disease symptoms as chlorosis or yellow streaks were recorded at the end of the stress period and pictures were taken. a Pictures of asymptomatic genome-edited plants (17, 76, and 81), symptomatic-edited plants (66 and 97), and wild type control plants (WT). b PCR diagnostic to detect activation of episomal BSOLV in genome edited and control plants under water stress conditions. c qPCR analysis to detect episomal BSOLV in genome edited and control plants under water stress conditions. PC1‒2, symptomatic plant of Agbagba from field as positive control; WT, wild type non-edited control Gonja Manjaya plant under stress conditions; 17, 66, 76, 81, 97, edited plants under stress conditions; CW, asymptomatic in vitro plantlet of Cavendish Williams as negative control; NTC no template control. For PCR and qPCR, leaf samples from three symptomatic wild type non-edited control plants (WT) of Gonja Manjaya were pooled for DNA extraction. Similarly, the leaves from three replicates for symptomatic and asymptomatic-edited events were pooled for PCR and qPCR. CT values were presented as means and standard error of six technical replicates from two independent experiments
    Figure Legend Snippet: Evaluation of genome-edited and wild type non-edited control plants of Gonja Manjaya for induction of BSV symptoms under water stress conditions. Two-month-old plants were subjected to water stress for 2 weeks. Disease symptoms as chlorosis or yellow streaks were recorded at the end of the stress period and pictures were taken. a Pictures of asymptomatic genome-edited plants (17, 76, and 81), symptomatic-edited plants (66 and 97), and wild type control plants (WT). b PCR diagnostic to detect activation of episomal BSOLV in genome edited and control plants under water stress conditions. c qPCR analysis to detect episomal BSOLV in genome edited and control plants under water stress conditions. PC1‒2, symptomatic plant of Agbagba from field as positive control; WT, wild type non-edited control Gonja Manjaya plant under stress conditions; 17, 66, 76, 81, 97, edited plants under stress conditions; CW, asymptomatic in vitro plantlet of Cavendish Williams as negative control; NTC no template control. For PCR and qPCR, leaf samples from three symptomatic wild type non-edited control plants (WT) of Gonja Manjaya were pooled for DNA extraction. Similarly, the leaves from three replicates for symptomatic and asymptomatic-edited events were pooled for PCR and qPCR. CT values were presented as means and standard error of six technical replicates from two independent experiments

    Techniques Used: Polymerase Chain Reaction, Diagnostic Assay, Activation Assay, Real-time Polymerase Chain Reaction, Positive Control, In Vitro, Negative Control, DNA Extraction

    39) Product Images from "CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes"

    Article Title: CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes

    Journal: bioRxiv

    doi: 10.1101/2020.03.29.015057

    tRNA insufficiency activates HIS4 transcription through a pathway that bypasses GCN2 kinase and eIF2a phosphorylation. ( A ) Comparison of P(HIS4) CiBER-seq profiles before and after 3AT treatment. ( B ) Schematic outlining observed ISR responses for functional categories of genes. ( C ) Western blot for eIF2α phosphorylation. Knockdown of amino acid biosynthesis ( ILV2 guide) and aminoacyl tRNA charging ( HTS1 guide) results in eIF2α phosphorylation while knockdown of RNA polymerase III ( RPC31 guide) does not. GCN2 deletion abolishes eIF2α phosphorylation. ( D ) Quantification of eIF2α phosphorylation relative to hexokinase loading control in (C). ( E ) Endogenous HIS4 mRNA levels measured by qPCR. Endogenous HIS4 activation by ILV2, HTS1, or RPC31 knockdown is completely GCN4 dependent. HIS4 activation by ILV2 knockdown is also completely GCN2 dependent while effects of RPC31 knockdown are entirely GCN2 independent and HTS1 knockdown is only partially GCN2 dependent.
    Figure Legend Snippet: tRNA insufficiency activates HIS4 transcription through a pathway that bypasses GCN2 kinase and eIF2a phosphorylation. ( A ) Comparison of P(HIS4) CiBER-seq profiles before and after 3AT treatment. ( B ) Schematic outlining observed ISR responses for functional categories of genes. ( C ) Western blot for eIF2α phosphorylation. Knockdown of amino acid biosynthesis ( ILV2 guide) and aminoacyl tRNA charging ( HTS1 guide) results in eIF2α phosphorylation while knockdown of RNA polymerase III ( RPC31 guide) does not. GCN2 deletion abolishes eIF2α phosphorylation. ( D ) Quantification of eIF2α phosphorylation relative to hexokinase loading control in (C). ( E ) Endogenous HIS4 mRNA levels measured by qPCR. Endogenous HIS4 activation by ILV2, HTS1, or RPC31 knockdown is completely GCN4 dependent. HIS4 activation by ILV2 knockdown is also completely GCN2 dependent while effects of RPC31 knockdown are entirely GCN2 independent and HTS1 knockdown is only partially GCN2 dependent.

    Techniques Used: Functional Assay, Western Blot, Real-time Polymerase Chain Reaction, Activation Assay

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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 probe universal one step 4x master mix
    Implementation and adaptation of the Saliva Direct RT-qPCR assay to detect SARS-CoV-2 N1 and human RNase P with the QuantStudio-6. a) Full and half reaction volumes on the CFx96 with 2x master mix, b) Full and half reaction volumes on the QuantStudio-6 with 2x master mix, c) Full and half reaction volumes on the QuantStudio-6 with <t>4x</t> master mix. Samples which failed to amplify are denoted as “not detected” (ND). Plasmid DNA was added directly into the master mix at 200,000 copies/mL (2-4x Saliva Direct limit of detection) in triplicate. Full reactions (15μL master mix with 5μL sample input), half reactions (7.5μL master mix with 5μL sample input).
    Luna Probe Universal One Step 4x Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, 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

    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

    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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0258263

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

    Article Snippet: Thank you to New England Biolabs for the gift of the Luna Probe Universal One-Step 4x master mix and technical support.

    Techniques: Quantitative RT-PCR, Plasmid Preparation

    Evaluating E-Sarbeco and RdRp-SARSr primers and probes with RNase P on the QuantStudio-6. a) Full and half-sized reactions of E-Sarbeco on the QuantStudio-6 with 4x master mix, b) Full and half-sized reactions of RdRp on the QuantStudio-6 with 4x master mix. Plasmid DNA was added directly into the master mix at 200,000 copies/mL (2-4x Saliva Direct limit of detection) in triplicate. Full reactions (15μL master mix with 5μL sample input), half reactions (7.5μL master mix with 5μL sample input). c) Evaluation of corrected RdRp reverse primers at two different concentrations on pooled RNA eluted from positive clinical swab samples in triplicate. p

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0258263

    Figure Lengend Snippet: Evaluating E-Sarbeco and RdRp-SARSr primers and probes with RNase P on the QuantStudio-6. a) Full and half-sized reactions of E-Sarbeco on the QuantStudio-6 with 4x master mix, b) Full and half-sized reactions of RdRp on the QuantStudio-6 with 4x master mix. Plasmid DNA was added directly into the master mix at 200,000 copies/mL (2-4x Saliva Direct limit of detection) in triplicate. Full reactions (15μL master mix with 5μL sample input), half reactions (7.5μL master mix with 5μL sample input). c) Evaluation of corrected RdRp reverse primers at two different concentrations on pooled RNA eluted from positive clinical swab samples in triplicate. p

    Article Snippet: Thank you to New England Biolabs for the gift of the Luna Probe Universal One-Step 4x master mix and technical support.

    Techniques: Plasmid Preparation