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 "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

    3) 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

    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

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    New England Biolabs luna universal qpcr master mix
    Generation of TUBL-deficient mice. (A) Schematic representation of the WT TINCR allele, the single-stranded oligodeoxynucleotide (ssODN), and the mutant allele after homologous recombination. Exons are denoted by numbered boxes. The single guide RNA (sgRNA) for the CRISPR-Cas9 system and its protospacer adjacent motif (PAM) are indicated by contiguous black and red underlines, respectively. The TUBL ORF is represented by the gray shading in the box corresponding to exon 1 of TINCR . (B) Predicted secondary structure and minimal free energy for WT TINCR and the mutant form generated by the CRISPR-Cas9 system for establishment of Tubl −/− mice. The triangle indicates the 5’ end of the transcript. (C) <t>PCR</t> analysis of genomic DNA from the tail of mice of the indicated genotypes. The PCR products were digested with EcoRI before electrophoresis. (D) <t>RT-qPCR</t> analysis of TINCR in the epidermis of Tubl +/+ and Tubl −/− mice. Data are means ± SD (n = 3 independent experiments). ***p
    Luna Universal Qpcr Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs luna universal one step rt qpcr kit
    Screening of compounds with antiviral activity targeting <t>SARS-CoV-2</t> host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by <t>RT-qPCR</t> (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p
    Luna Universal One Step Rt Qpcr Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Generation of TUBL-deficient mice. (A) Schematic representation of the WT TINCR allele, the single-stranded oligodeoxynucleotide (ssODN), and the mutant allele after homologous recombination. Exons are denoted by numbered boxes. The single guide RNA (sgRNA) for the CRISPR-Cas9 system and its protospacer adjacent motif (PAM) are indicated by contiguous black and red underlines, respectively. The TUBL ORF is represented by the gray shading in the box corresponding to exon 1 of TINCR . (B) Predicted secondary structure and minimal free energy for WT TINCR and the mutant form generated by the CRISPR-Cas9 system for establishment of Tubl −/− mice. The triangle indicates the 5’ end of the transcript. (C) PCR analysis of genomic DNA from the tail of mice of the indicated genotypes. The PCR products were digested with EcoRI before electrophoresis. (D) RT-qPCR analysis of TINCR in the epidermis of Tubl +/+ and Tubl −/− mice. Data are means ± SD (n = 3 independent experiments). ***p

    Journal: PLoS Genetics

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

    doi: 10.1371/journal.pgen.1009686

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

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

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

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

    Journal: bioRxiv

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

    doi: 10.1101/2022.05.20.492787

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

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

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

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

    Journal: bioRxiv

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

    doi: 10.1101/2022.05.20.492787

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

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

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

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

    Journal: bioRxiv

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

    doi: 10.1101/2022.05.20.492787

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

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

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

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

    Journal: bioRxiv

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

    doi: 10.1101/2022.05.20.492787

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

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

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

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

    Journal: bioRxiv

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

    doi: 10.1101/2021.03.23.436611

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

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

    Techniques: Activity Assay, Infection, Quantitative RT-PCR

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

    Journal: bioRxiv

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

    doi: 10.1101/2021.03.23.436611

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

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

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

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

    Journal: Frontiers in Cell and Developmental Biology

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

    doi: 10.3389/fcell.2022.855340

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

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

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

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

    Journal: Frontiers in Cell and Developmental Biology

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

    doi: 10.3389/fcell.2022.855340

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

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

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

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

    Journal: Frontiers in Cell and Developmental Biology

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

    doi: 10.3389/fcell.2022.855340

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

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

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