hot firepol evagreen qpcr mix  (Solis BioDyne)


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    Solis BioDyne hot firepol evagreen qpcr mix
    Intraperitoneal administration of ethidium bromide activates UPR mt in muscle. Balb/c mices were injected though the intraperitoneal route, with 2 different concentration of ethidium bromide (10 mg/kg; 50 mg/kg) and methacycline hydrochloride (100 mg/kg; 200 mg/kg). The quadriceps muscle from mouse hind limb was isolated after 16 hrs of drug administration. The muscle RNA was isolated and expression of mitochondrial specific chaperones and protease (hspd1, hsp10, hsp75 and clpp1) were measured using <t>QPCR.</t> The bars show the average ± SEM of 3 mice per group (***: p
    Hot Firepol Evagreen Qpcr Mix, supplied by Solis BioDyne, 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 chemical screen to identify inducers of the mitochondrial unfolded protein response in C. elegans"

    Article Title: A chemical screen to identify inducers of the mitochondrial unfolded protein response in C. elegans

    Journal: Worm

    doi: 10.1080/21624054.2015.1096490

    Intraperitoneal administration of ethidium bromide activates UPR mt in muscle. Balb/c mices were injected though the intraperitoneal route, with 2 different concentration of ethidium bromide (10 mg/kg; 50 mg/kg) and methacycline hydrochloride (100 mg/kg; 200 mg/kg). The quadriceps muscle from mouse hind limb was isolated after 16 hrs of drug administration. The muscle RNA was isolated and expression of mitochondrial specific chaperones and protease (hspd1, hsp10, hsp75 and clpp1) were measured using QPCR. The bars show the average ± SEM of 3 mice per group (***: p
    Figure Legend Snippet: Intraperitoneal administration of ethidium bromide activates UPR mt in muscle. Balb/c mices were injected though the intraperitoneal route, with 2 different concentration of ethidium bromide (10 mg/kg; 50 mg/kg) and methacycline hydrochloride (100 mg/kg; 200 mg/kg). The quadriceps muscle from mouse hind limb was isolated after 16 hrs of drug administration. The muscle RNA was isolated and expression of mitochondrial specific chaperones and protease (hspd1, hsp10, hsp75 and clpp1) were measured using QPCR. The bars show the average ± SEM of 3 mice per group (***: p

    Techniques Used: Injection, Concentration Assay, Isolation, Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

    2) Product Images from "The zebrafish orthologue of the human hepatocerebral disease gene MPV17 plays pleiotropic roles in mitochondria"

    Article Title: The zebrafish orthologue of the human hepatocerebral disease gene MPV17 plays pleiotropic roles in mitochondria

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.037226

    Biochemical and genetic characterization of mitochondrial phenotype of  mpv17  KO larvae.  (A) Basal OCR was measured for ∼1 h in fish water, immediately after 4 dpf larvae were exposed to 0.5 μM FCCP and, later, to a combination of 2 μM rotenone (Rot) and 5 μM antimycin (AA). Four independent experiments were performed ( n =40). (B) Quantification of basal respiration in wild-type and  mpv17 −/−  mutants ( n =40). (C) Protein blot analysis of different subunits of OXPHOS complexes. (D) Relative quantification of protein amount, using an antibody against βActin for standardization ( n =3). R.I., relative intensity. (E) Relative quantification of mtDNA copy number in wild type and  mpv17  homozygous mutants. Mean dCt values were calculated as Ct of  mt-nd1  (mitochondrially encoded gene) minus Ct of  polg  (nuclear gene) and plotted with s.e.m. ( n =7). (F) Real-time PCR quantification of mRNA transcripts from Ubiquinol-cytochrome c reductase complex subunits ( n =6). Statistical analyses were performed using two-tailed Student's  t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. **** P
    Figure Legend Snippet: Biochemical and genetic characterization of mitochondrial phenotype of mpv17 KO larvae. (A) Basal OCR was measured for ∼1 h in fish water, immediately after 4 dpf larvae were exposed to 0.5 μM FCCP and, later, to a combination of 2 μM rotenone (Rot) and 5 μM antimycin (AA). Four independent experiments were performed ( n =40). (B) Quantification of basal respiration in wild-type and mpv17 −/− mutants ( n =40). (C) Protein blot analysis of different subunits of OXPHOS complexes. (D) Relative quantification of protein amount, using an antibody against βActin for standardization ( n =3). R.I., relative intensity. (E) Relative quantification of mtDNA copy number in wild type and mpv17 homozygous mutants. Mean dCt values were calculated as Ct of mt-nd1 (mitochondrially encoded gene) minus Ct of polg (nuclear gene) and plotted with s.e.m. ( n =7). (F) Real-time PCR quantification of mRNA transcripts from Ubiquinol-cytochrome c reductase complex subunits ( n =6). Statistical analyses were performed using two-tailed Student's t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. **** P

    Techniques Used: Fluorescence In Situ Hybridization, Real-time Polymerase Chain Reaction, Two Tailed Test

    Evaluation of stress response and mitochondrial quality control system in zebrafish larvae.  (A) Representative western blot analysis of Grp75 protein from 6 dpf zebrafish larvae and relative quantification, using an antibody against Tomm20 (also known as Tomm20b) for standardization ( n =3). (B) Real-time PCR quantification of mRNA transcripts from different MICOS subunits at 6 dpf ( n =8). Statistical analyses were performed using two-tailed Student's  t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. *** P
    Figure Legend Snippet: Evaluation of stress response and mitochondrial quality control system in zebrafish larvae. (A) Representative western blot analysis of Grp75 protein from 6 dpf zebrafish larvae and relative quantification, using an antibody against Tomm20 (also known as Tomm20b) for standardization ( n =3). (B) Real-time PCR quantification of mRNA transcripts from different MICOS subunits at 6 dpf ( n =8). Statistical analyses were performed using two-tailed Student's t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. *** P

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

    Investigation of  mpv17  orthologue and paralogue genes in zebrafish larvae.  (A) Real-time PCR quantification of mRNA transcripts of  mpv17-like2  and  mpv17-like  at 6 dpf ( n =9). (B) Evaluation of phenotypic rescue in the tail region at 3 dpf after the injection of human  MPV17  mRNAs, wild-type (WT) and p.R50Q mutated forms, and zebrafish  mpv17  and  mpv17-like2  mRNAs. Arrowheads point to iridophores. Scale bars: 100 µm. (C) Relative quantification of iridophore amount in controls and injected larvae ( n =30). (D) mtDNA copy number analysis in  mpv17 −/−  mutants transiently overexpressing  mpv17-like2  at 3 dpf and 6 dpf. Mean dCt values were calculated as Ct of  mt-nd1  (mitochondrially encoded gene) minus Ct of  polg  (nuclear gene) ( n =4). Statistical analyses were performed using two-tailed Student's  t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. **** P
    Figure Legend Snippet: Investigation of mpv17 orthologue and paralogue genes in zebrafish larvae. (A) Real-time PCR quantification of mRNA transcripts of mpv17-like2 and mpv17-like at 6 dpf ( n =9). (B) Evaluation of phenotypic rescue in the tail region at 3 dpf after the injection of human MPV17 mRNAs, wild-type (WT) and p.R50Q mutated forms, and zebrafish mpv17 and mpv17-like2 mRNAs. Arrowheads point to iridophores. Scale bars: 100 µm. (C) Relative quantification of iridophore amount in controls and injected larvae ( n =30). (D) mtDNA copy number analysis in mpv17 −/− mutants transiently overexpressing mpv17-like2 at 3 dpf and 6 dpf. Mean dCt values were calculated as Ct of mt-nd1 (mitochondrially encoded gene) minus Ct of polg (nuclear gene) ( n =4). Statistical analyses were performed using two-tailed Student's t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. **** P

    Techniques Used: Real-time Polymerase Chain Reaction, Injection, Two Tailed Test

    3) Product Images from "Capped nascent RNA sequencing reveals novel therapy-responsive enhancers in prostate cancer"

    Article Title: Capped nascent RNA sequencing reveals novel therapy-responsive enhancers in prostate cancer

    Journal: bioRxiv

    doi: 10.1101/2022.04.08.487666

    Functional validation of PRO-cap enhancers. A) Comparing changes in eRNA expression with ENZ treatment as detected by PRO-cap with changes in H3K27Ac ChIP-seq and ATAC-seq. XY charts and heatmaps show read density 1kb up- and downstream of the center of the ENZ regulated enhancers. Data is from publicly available data 57 (GSE137775). B) Selected candidate enhancers for functional analysis. C) Analysis of changes in enhancer activity with androgen stimulation or inhibition as determined by luciferase reported assays does not agree with PRO-cap data. 24 hours post transfection cells were treated with 10 μM enzalutamide or 10 nM DHT for and additional 24 hours. Shown is the average log2 fold change between DMSO and ENZ or EtOH and DHT from n = 3 experiments, each with n = 3 replicates. Statistics were determined by t-test. D) Analysis of eRNA expression in response to AR stimulation (10 nM DHT) or inhibition (10 μM ENZ). Expression was measured via qPCR (n = 3) and is displayed as the average log2 fold change with treatment. Statistics were determined by t-test . E) Categorization of ENZ-regulated enhancers identified with PRO-cap in publish STARR-seq data 31 . Activity of 139 of the 853 candidate enhancers was measured in response to androgen stimulation (DHT) and categorized as inactive (no enhancer activity), constitutively active (no change in activity with DHT) or induced by DHT. Statistics were determined using Fisher’s exact test.
    Figure Legend Snippet: Functional validation of PRO-cap enhancers. A) Comparing changes in eRNA expression with ENZ treatment as detected by PRO-cap with changes in H3K27Ac ChIP-seq and ATAC-seq. XY charts and heatmaps show read density 1kb up- and downstream of the center of the ENZ regulated enhancers. Data is from publicly available data 57 (GSE137775). B) Selected candidate enhancers for functional analysis. C) Analysis of changes in enhancer activity with androgen stimulation or inhibition as determined by luciferase reported assays does not agree with PRO-cap data. 24 hours post transfection cells were treated with 10 μM enzalutamide or 10 nM DHT for and additional 24 hours. Shown is the average log2 fold change between DMSO and ENZ or EtOH and DHT from n = 3 experiments, each with n = 3 replicates. Statistics were determined by t-test. D) Analysis of eRNA expression in response to AR stimulation (10 nM DHT) or inhibition (10 μM ENZ). Expression was measured via qPCR (n = 3) and is displayed as the average log2 fold change with treatment. Statistics were determined by t-test . E) Categorization of ENZ-regulated enhancers identified with PRO-cap in publish STARR-seq data 31 . Activity of 139 of the 853 candidate enhancers was measured in response to androgen stimulation (DHT) and categorized as inactive (no enhancer activity), constitutively active (no change in activity with DHT) or induced by DHT. Statistics were determined using Fisher’s exact test.

    Techniques Used: Functional Assay, Expressing, Chromatin Immunoprecipitation, Activity Assay, Inhibition, Luciferase, Transfection, Real-time Polymerase Chain Reaction

    4) Product Images from "Phorbol ester-mediated re-expression of endogenous LAT adapter in J.CaM2 cells: a model for dissecting drivers and blockers of LAT transcription"

    Article Title: Phorbol ester-mediated re-expression of endogenous LAT adapter in J.CaM2 cells: a model for dissecting drivers and blockers of LAT transcription

    Journal: Genes and Immunity

    doi: 10.1038/gene.2016.25

    Identification of two point mutations in LAT gene of J.CaM2 cells by DNA sequencing and PCR–RFLP. ( a ) Left panel: histograms showing homozygous wild type 'C' nucleotide in intron 1 at the position g.237 in Jurkat and heterozygous variant C > T found in J.CaM2. The mutation was further confirmed by the digestion of PCR product (amplified from genomic DNA) with Bam HI endonuclease. Right panel: histogram of homozygous, wild type 'C' base in exon 4 at position c.167 in Jurkat and heterozygous c.167C > T missense mutation in J.CaM2 changing threonine to methionine at position 56. Presence of the variant was verified by the digestion of PCR product (generated using cDNA) with Nla III. ( b ) Scheme of the pGL4.14– LAT (−916/+357) plasmids used to test the effect of g.237C > T mutation on LAT promoter activity in Jurkat cells under resting and activation conditions. The results of dual-luciferase assay are representative of four technical replicates that represent two independent experiments ( P
    Figure Legend Snippet: Identification of two point mutations in LAT gene of J.CaM2 cells by DNA sequencing and PCR–RFLP. ( a ) Left panel: histograms showing homozygous wild type 'C' nucleotide in intron 1 at the position g.237 in Jurkat and heterozygous variant C > T found in J.CaM2. The mutation was further confirmed by the digestion of PCR product (amplified from genomic DNA) with Bam HI endonuclease. Right panel: histogram of homozygous, wild type 'C' base in exon 4 at position c.167 in Jurkat and heterozygous c.167C > T missense mutation in J.CaM2 changing threonine to methionine at position 56. Presence of the variant was verified by the digestion of PCR product (generated using cDNA) with Nla III. ( b ) Scheme of the pGL4.14– LAT (−916/+357) plasmids used to test the effect of g.237C > T mutation on LAT promoter activity in Jurkat cells under resting and activation conditions. The results of dual-luciferase assay are representative of four technical replicates that represent two independent experiments ( P

    Techniques Used: DNA Sequencing, Polymerase Chain Reaction, Variant Assay, Mutagenesis, Amplification, Generated, Activity Assay, Activation Assay, Luciferase

    Histone modifications and recruitment of Sp transcription factors associated with the steady-state and PMA-induced expression of LAT. ( a ) ChIP-seq profiles derived from publicly available ChIP-seq data sets for RNA Polymerase II (Pol II; GSE50622), DNase I hypersensitive sites (DHS; GSE29692), H3K4me3 (GSE35583) and H3K27ac (GSE59257)—visualized for LAT gene in resting Jurkat cells. The region defined earlier using dual-reporter assay 10 as LAT proximal promoter (P) is bound by Pol II and displays DNase I hypersensitivity. The promoter is also occupied by H3K4me3 and H3K27ac that are indicative of active promoters. ( b ) Representative ChIP–PCR results revealing binding signals of H3K27ac, Sp1 and Sp3 on chosen LAT gene regions in untreated and PMA stimulated J.CaM2 cells. The experiment was repeated four times giving reproducible results.
    Figure Legend Snippet: Histone modifications and recruitment of Sp transcription factors associated with the steady-state and PMA-induced expression of LAT. ( a ) ChIP-seq profiles derived from publicly available ChIP-seq data sets for RNA Polymerase II (Pol II; GSE50622), DNase I hypersensitive sites (DHS; GSE29692), H3K4me3 (GSE35583) and H3K27ac (GSE59257)—visualized for LAT gene in resting Jurkat cells. The region defined earlier using dual-reporter assay 10 as LAT proximal promoter (P) is bound by Pol II and displays DNase I hypersensitivity. The promoter is also occupied by H3K4me3 and H3K27ac that are indicative of active promoters. ( b ) Representative ChIP–PCR results revealing binding signals of H3K27ac, Sp1 and Sp3 on chosen LAT gene regions in untreated and PMA stimulated J.CaM2 cells. The experiment was repeated four times giving reproducible results.

    Techniques Used: Expressing, Chromatin Immunoprecipitation, Derivative Assay, Reporter Assay, Polymerase Chain Reaction, Binding Assay

    5) Product Images from "A robust (re-)annotation approach to generate unbiased mapping references for RNA-seq-based analyses of differential expression across closely related species"

    Article Title: A robust (re-)annotation approach to generate unbiased mapping references for RNA-seq-based analyses of differential expression across closely related species

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2646-x

    qPCR results . Boxplot of normalized Ct values (reference gene: actin 79B ) For each studied gene (one colour) boxplot is showed for Ct values in D. melanogaster OreR (“D. mel”) and D. mauritiana TAM16 (“D. mau”). (Significance calculated by t-test (for genes with homogeneous distribution of variances) or t-Welch-test (for genes with not homogeneous distribution of variances); * p
    Figure Legend Snippet: qPCR results . Boxplot of normalized Ct values (reference gene: actin 79B ) For each studied gene (one colour) boxplot is showed for Ct values in D. melanogaster OreR (“D. mel”) and D. mauritiana TAM16 (“D. mau”). (Significance calculated by t-test (for genes with homogeneous distribution of variances) or t-Welch-test (for genes with not homogeneous distribution of variances); * p

    Techniques Used: Real-time Polymerase Chain Reaction

    6) Product Images from "Pseudomonas aeruginosa partitioning protein ParB acts as a nucleoid-associated protein binding to multiple copies of a parS-related motif"

    Article Title: Pseudomonas aeruginosa partitioning protein ParB acts as a nucleoid-associated protein binding to multiple copies of a parS-related motif

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky257

    ParB binding to parS sites in Pseudomonas aeruginosa genome. ( A ) Western blot analysis of elution fractions from DNA pull-down assays performed with DNA fragments containing parS1–parS10 sequences. Biotinylated DNA was coupled with magnetic beads, incubated with extracts of PAO1161 cells and washed. Proteins bound to the DNA were eluted with increasing concentration of NaCl (0.2–1 M). The negative control contained water instead of biotinylated DNA. Western blotting was performed using polyclonal anti-ParB antibodies. ( B–G ) qPCR analysis of DNA obtained by nucleoprotein immunoprecipitation using anti-ParB antibodies and extracts from exponentially growing cells of P. aeruginosa strains (B) WT, (C) ParB+++, (D) parS2 + , (E) parS1-4 , (F) parS null and (G) parB null (negative control). qPCR was performed using primers flanking the parS sequences and for the proC gene (background control). Data represent percentage of DNA recovered by ChIP relative to corresponding input samples and are shown as mean ±SD for at least three biological replicates analyzed in three technical replicates. The significance of differences in recovery of parS s relative to the background control ( proC ) was evaluated by two-sided Student’s t -test assuming equal variance, with P
    Figure Legend Snippet: ParB binding to parS sites in Pseudomonas aeruginosa genome. ( A ) Western blot analysis of elution fractions from DNA pull-down assays performed with DNA fragments containing parS1–parS10 sequences. Biotinylated DNA was coupled with magnetic beads, incubated with extracts of PAO1161 cells and washed. Proteins bound to the DNA were eluted with increasing concentration of NaCl (0.2–1 M). The negative control contained water instead of biotinylated DNA. Western blotting was performed using polyclonal anti-ParB antibodies. ( B–G ) qPCR analysis of DNA obtained by nucleoprotein immunoprecipitation using anti-ParB antibodies and extracts from exponentially growing cells of P. aeruginosa strains (B) WT, (C) ParB+++, (D) parS2 + , (E) parS1-4 , (F) parS null and (G) parB null (negative control). qPCR was performed using primers flanking the parS sequences and for the proC gene (background control). Data represent percentage of DNA recovered by ChIP relative to corresponding input samples and are shown as mean ±SD for at least three biological replicates analyzed in three technical replicates. The significance of differences in recovery of parS s relative to the background control ( proC ) was evaluated by two-sided Student’s t -test assuming equal variance, with P

    Techniques Used: Binding Assay, Western Blot, Magnetic Beads, Incubation, Concentration Assay, Negative Control, Real-time Polymerase Chain Reaction, Immunoprecipitation, Chromatin Immunoprecipitation

    7) Product Images from "Quantitative PCR provides a simple and accessible method for quantitative microbiota profiling"

    Article Title: Quantitative PCR provides a simple and accessible method for quantitative microbiota profiling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0227285

    Relative microbiome profiles translated into quantitative microbiome profiles using qPCR. (a) Comparison of relative abundances and estimated absolute abundances of dominant bacterial families in 114 fecal samples. The top panel shows relative abundances based on 16S amplicon sequencing and the lower panels shows the estimated absolute abundances calculated by multiplying the relative abundances with total bacterial load, i.e. qPCR-based estimate of copies of 16S gene per 1 g of feces. (b) Correlation between the qPCR abundances (16S rRNA gene copies per g feces) and the estimated absolute abundances of four taxa representing species, genus, family and phylum levels. The dashed line shows the expected 1:1 correspondence. The correspondence decreases at the very low end of the abundance range, likely due to the relatively lower PCR amplification efficiency and increased stochasticity of the results for low abundance taxa in NGS [ 30 ]. The applied library preparation method (dual index TruSeq-tailed 1-step amplification [ 17 ]) causes a slight underestimation of Bacteroidetes abundance (unpublished data), explaining the underestimation observed for this phylum compared to qPCR. (c) and (d) show the associations between the qPCR-determined abundance of the butyryl-CoA:acetate CoA-transferase gene and the (c) estimated absolute abundance and (d) relative abundance of butyrate producers detected in the NGS data.
    Figure Legend Snippet: Relative microbiome profiles translated into quantitative microbiome profiles using qPCR. (a) Comparison of relative abundances and estimated absolute abundances of dominant bacterial families in 114 fecal samples. The top panel shows relative abundances based on 16S amplicon sequencing and the lower panels shows the estimated absolute abundances calculated by multiplying the relative abundances with total bacterial load, i.e. qPCR-based estimate of copies of 16S gene per 1 g of feces. (b) Correlation between the qPCR abundances (16S rRNA gene copies per g feces) and the estimated absolute abundances of four taxa representing species, genus, family and phylum levels. The dashed line shows the expected 1:1 correspondence. The correspondence decreases at the very low end of the abundance range, likely due to the relatively lower PCR amplification efficiency and increased stochasticity of the results for low abundance taxa in NGS [ 30 ]. The applied library preparation method (dual index TruSeq-tailed 1-step amplification [ 17 ]) causes a slight underestimation of Bacteroidetes abundance (unpublished data), explaining the underestimation observed for this phylum compared to qPCR. (c) and (d) show the associations between the qPCR-determined abundance of the butyryl-CoA:acetate CoA-transferase gene and the (c) estimated absolute abundance and (d) relative abundance of butyrate producers detected in the NGS data.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Sequencing, Polymerase Chain Reaction, Next-Generation Sequencing

    Workflow for implementation of qPCR-based quantitative microbiome profiling. All of these steps are included in R package mare [ 18 ].
    Figure Legend Snippet: Workflow for implementation of qPCR-based quantitative microbiome profiling. All of these steps are included in R package mare [ 18 ].

    Techniques Used: Real-time Polymerase Chain Reaction

    8) Product Images from "DNA Topoisomerases Are Required for Preinitiation Complex Assembly during GAL Gene Activation"

    Article Title: DNA Topoisomerases Are Required for Preinitiation Complex Assembly during GAL Gene Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0132739

    GAL gene transcription requires topoisomerase activity. (A) Organization of the GAL genes. GAL1 and GAL10 are located on Chromosome II (Chr. II) and share a promoter with two UASs and three nucleosomes. The GAL7 promoter, located immediately after the GAL10 open reading frame on Chr. II, contains a single UAS and a single nucleosome. The GAL2 promoter is similar to the GAL7 promoter, but is located on Chr. XII. Promoter nucleosomes are illustrated in yellow. A promoter nucleosome occupancy profile [ 6 ] is shown below each gene (black line) indicating chromosomal coordinates of nucleosomes. Primers used in ChIP experiments are indicated by black arrows. UAS, Upstream Activating Sequence. TATA, TATA box. (B) Experimental setup. Cells were grown at 25°C in raffinose media (de-repressive conditions), and α-factor was added to arrest cells in G1. After 1.5 hours, cells were shifted to the restrictive temperature. After Top2 inactivation for 15 minutes, cells were treated with galactose to induce the GAL genes, α-factor was added again to keep cells in G1, and samples were collected at the indicated time points. α, α-factor. (C) Induction of GAL1 , GAL2 , GAL7 , and GAL10 in wild type and top1Δtop2 ts cells. Cells were treated as illustrated in (B), and samples were collected 0 and 150 minutes after galactose treatment. mRNA was isolated, and the levels of the individual GAL genes and two control genes ( GAPDH and ACT1 ) were quantified by qPCR. mRNA levels of the GAL genes were calculated relative to the mean of the mRNA levels for GAPDH and ACT1 for each time point and the value obtained in wild type at the latest time point was set to 100. Numbers indicate relative mRNA levels. Averages from two individual experiments are shown with error bars representing ± one standard deviation. (D) Induction of GAL1 , GAL2 , GAL7 , and GAL10 in wild type, top1Δ , top2 ts , and top1Δtop2 ts cells. Cells were treated as shown in (B), and samples were collected at the indicated time points for qPCR measurements of GAL gene mRNA levels. mRNA levels are presented as fold increase relative to the level at time point 0. Averages from three individual experiments are shown with error bars representing ± one standard deviation.
    Figure Legend Snippet: GAL gene transcription requires topoisomerase activity. (A) Organization of the GAL genes. GAL1 and GAL10 are located on Chromosome II (Chr. II) and share a promoter with two UASs and three nucleosomes. The GAL7 promoter, located immediately after the GAL10 open reading frame on Chr. II, contains a single UAS and a single nucleosome. The GAL2 promoter is similar to the GAL7 promoter, but is located on Chr. XII. Promoter nucleosomes are illustrated in yellow. A promoter nucleosome occupancy profile [ 6 ] is shown below each gene (black line) indicating chromosomal coordinates of nucleosomes. Primers used in ChIP experiments are indicated by black arrows. UAS, Upstream Activating Sequence. TATA, TATA box. (B) Experimental setup. Cells were grown at 25°C in raffinose media (de-repressive conditions), and α-factor was added to arrest cells in G1. After 1.5 hours, cells were shifted to the restrictive temperature. After Top2 inactivation for 15 minutes, cells were treated with galactose to induce the GAL genes, α-factor was added again to keep cells in G1, and samples were collected at the indicated time points. α, α-factor. (C) Induction of GAL1 , GAL2 , GAL7 , and GAL10 in wild type and top1Δtop2 ts cells. Cells were treated as illustrated in (B), and samples were collected 0 and 150 minutes after galactose treatment. mRNA was isolated, and the levels of the individual GAL genes and two control genes ( GAPDH and ACT1 ) were quantified by qPCR. mRNA levels of the GAL genes were calculated relative to the mean of the mRNA levels for GAPDH and ACT1 for each time point and the value obtained in wild type at the latest time point was set to 100. Numbers indicate relative mRNA levels. Averages from two individual experiments are shown with error bars representing ± one standard deviation. (D) Induction of GAL1 , GAL2 , GAL7 , and GAL10 in wild type, top1Δ , top2 ts , and top1Δtop2 ts cells. Cells were treated as shown in (B), and samples were collected at the indicated time points for qPCR measurements of GAL gene mRNA levels. mRNA levels are presented as fold increase relative to the level at time point 0. Averages from three individual experiments are shown with error bars representing ± one standard deviation.

    Techniques Used: Activity Assay, Chromatin Immunoprecipitation, Sequencing, Isolation, Real-time Polymerase Chain Reaction, Standard Deviation

    Topoisomerase activity has a direct role in GAL gene activation but is not required for transcriptional elongation and reinitiation. (A) Overview of promoter changes during transcriptional induction of the GAL genes. ( left ) In raffinose, the GAL gene promoter is covered by nucleosomes except at the UAS, which binds Gal4 having its activation domain blocked by Gal80. ( right ) Upon galactose addition, Gal3 binds Gal80, leaving the activation domain of Gal4 free to bind chromatin remodelers. Subsequent removal of promoter nucleosomes allows recruitment of TBP and RNA polymerase II. UAS, Upstream Activating Sequence. TATA, TATA box. TSS, Transcription Start Site. gal, galactose. Light coloring and dashed borders of Gal3 and Gal80 indicate that the enzymes do not block the Gal4 activation domain, either due to dissociation or rearrangement of the complex. (B) Time course experiment of GAL1 , GAL2 , GAL7 , and GAL10 transcription in wild type, gal80Δ , and gal80Δtop1Δtop2 ts cells. Cells were treated as in Fig 1B , and mRNA levels of the individual genes were quantified by qPCR, normalized to the wild type level at the latest time point (set to 100%), and presented on a log10-scale. The average from two individual experiments is shown, and error bars represent ± one standard deviation. (C) Time course experiment with ChIP analysis of RNA polymerase II enrichment in the coding regions of the GAL genes and two control genes, GAPDH and ACTI , following transcriptional activation. Cells were treated as in Fig 1B , and ChIP was performed with antibodies recognizing the C-terminal domain of the Rpb1 subunit of RNA polymerase II. RNA polymerase II binding levels were normalized relative to the binding at the 0 min time point (set to 1). (D) Time course experiment with ChIP analysis of Top1 and Top2 enrichment in the promoters of the GAL genes following transcriptional activation. Cells expressing the endogenous Top1 or Top2 enzymes fused to a cMyc tag were treated as described in Fig 1B , and ChIP was performed with antibodies recognizing the cMyc tag. Top1 and Top2 binding levels were normalized as in (C). In (C) and (D) averages from three individual experiments are shown, and error bars represent ± one standard deviation. Positions of primers used in the ChIP experiments for the individual GAL genes are indicated with arrows in Fig 1A and presented in Table 2 .
    Figure Legend Snippet: Topoisomerase activity has a direct role in GAL gene activation but is not required for transcriptional elongation and reinitiation. (A) Overview of promoter changes during transcriptional induction of the GAL genes. ( left ) In raffinose, the GAL gene promoter is covered by nucleosomes except at the UAS, which binds Gal4 having its activation domain blocked by Gal80. ( right ) Upon galactose addition, Gal3 binds Gal80, leaving the activation domain of Gal4 free to bind chromatin remodelers. Subsequent removal of promoter nucleosomes allows recruitment of TBP and RNA polymerase II. UAS, Upstream Activating Sequence. TATA, TATA box. TSS, Transcription Start Site. gal, galactose. Light coloring and dashed borders of Gal3 and Gal80 indicate that the enzymes do not block the Gal4 activation domain, either due to dissociation or rearrangement of the complex. (B) Time course experiment of GAL1 , GAL2 , GAL7 , and GAL10 transcription in wild type, gal80Δ , and gal80Δtop1Δtop2 ts cells. Cells were treated as in Fig 1B , and mRNA levels of the individual genes were quantified by qPCR, normalized to the wild type level at the latest time point (set to 100%), and presented on a log10-scale. The average from two individual experiments is shown, and error bars represent ± one standard deviation. (C) Time course experiment with ChIP analysis of RNA polymerase II enrichment in the coding regions of the GAL genes and two control genes, GAPDH and ACTI , following transcriptional activation. Cells were treated as in Fig 1B , and ChIP was performed with antibodies recognizing the C-terminal domain of the Rpb1 subunit of RNA polymerase II. RNA polymerase II binding levels were normalized relative to the binding at the 0 min time point (set to 1). (D) Time course experiment with ChIP analysis of Top1 and Top2 enrichment in the promoters of the GAL genes following transcriptional activation. Cells expressing the endogenous Top1 or Top2 enzymes fused to a cMyc tag were treated as described in Fig 1B , and ChIP was performed with antibodies recognizing the cMyc tag. Top1 and Top2 binding levels were normalized as in (C). In (C) and (D) averages from three individual experiments are shown, and error bars represent ± one standard deviation. Positions of primers used in the ChIP experiments for the individual GAL genes are indicated with arrows in Fig 1A and presented in Table 2 .

    Techniques Used: Activity Assay, Activation Assay, Sequencing, Blocking Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Chromatin Immunoprecipitation, Binding Assay, Expressing

    9) Product Images from "Gene activation by dCas9-CBP and the SAM system differ in target preference"

    Article Title: Gene activation by dCas9-CBP and the SAM system differ in target preference

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-54179-x

    Direct fusion of the CBP HAT domain to dCas9 outperforms a MS2 coat protein-CBP fusion plus dCas9 combination. ( A ) Schematic drawings of dCas9 fused to the HAT domain of CBP, MS2 coat protein fused to the CBP HAT domain (MCP-CBP) where two MCP dimers recognize two MS2 loops in the gRNA and dCas9 thereby brings four CBP domains to the locus, dCas9-VPR where three activation domains are fused to dCas9, and the synergistic activation mediator (SAM) system where MCP targets eight activation domains to dCas9 fused with the VP64 activation domain. ( B ) RT-qPCR showing twist ( twi ) expression in Drosophila S2 cells and in S2 cells transfected with UAS-dCas9 fusions or UAS-dCas9 and UAS-MCP fusions together with Actin -Gal4 in the presence of a control gRNA or twi promoter gRNA. Expression is plotted relative to RP49 . n = 3 biological replicates and error bars represent S.E.M. One-way ANOVA with post hoc Tukey test was used to calculate statistically significant differences. The full statistical analysis and fold activation in relation to the control (QUAS) gRNA is shown in Supplementary Table 2 . The F2161A mutation disrupts the catalytic function of the CBP HAT domain. Below the graph is a schematic drawing of the twi locus. ( C ) Western blot showing expression of the dCas9 and MCP fusion proteins. Uncropped images are shown in Supplemental Fig. S9 .
    Figure Legend Snippet: Direct fusion of the CBP HAT domain to dCas9 outperforms a MS2 coat protein-CBP fusion plus dCas9 combination. ( A ) Schematic drawings of dCas9 fused to the HAT domain of CBP, MS2 coat protein fused to the CBP HAT domain (MCP-CBP) where two MCP dimers recognize two MS2 loops in the gRNA and dCas9 thereby brings four CBP domains to the locus, dCas9-VPR where three activation domains are fused to dCas9, and the synergistic activation mediator (SAM) system where MCP targets eight activation domains to dCas9 fused with the VP64 activation domain. ( B ) RT-qPCR showing twist ( twi ) expression in Drosophila S2 cells and in S2 cells transfected with UAS-dCas9 fusions or UAS-dCas9 and UAS-MCP fusions together with Actin -Gal4 in the presence of a control gRNA or twi promoter gRNA. Expression is plotted relative to RP49 . n = 3 biological replicates and error bars represent S.E.M. One-way ANOVA with post hoc Tukey test was used to calculate statistically significant differences. The full statistical analysis and fold activation in relation to the control (QUAS) gRNA is shown in Supplementary Table 2 . The F2161A mutation disrupts the catalytic function of the CBP HAT domain. Below the graph is a schematic drawing of the twi locus. ( C ) Western blot showing expression of the dCas9 and MCP fusion proteins. Uncropped images are shown in Supplemental Fig. S9 .

    Techniques Used: HAT Assay, Activation Assay, Quantitative RT-PCR, Expressing, Transfection, Mutagenesis, Western Blot

    10) Product Images from "Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages"

    Article Title: Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0229-y

    Effect of dynasore hydrate on the SW480 and SW620 EV-induced changes on the expression of the surface marker CD14 and on the gene expression of CXCL10 and IL-10 in M0 macrophages. a Flow cytometry analysis showing the percentage of CD14-positive M0 macrophages. The graphs represent mean ± SD (n = 2). Statistical analysis was carried out with t-test. * p ≤ 0.05, ** p ≤ 0.01 b qPCR analysis showing changes in CXCL10 and IL-10 gene expression (n = 3).
    Figure Legend Snippet: Effect of dynasore hydrate on the SW480 and SW620 EV-induced changes on the expression of the surface marker CD14 and on the gene expression of CXCL10 and IL-10 in M0 macrophages. a Flow cytometry analysis showing the percentage of CD14-positive M0 macrophages. The graphs represent mean ± SD (n = 2). Statistical analysis was carried out with t-test. * p ≤ 0.05, ** p ≤ 0.01 b qPCR analysis showing changes in CXCL10 and IL-10 gene expression (n = 3).

    Techniques Used: Expressing, Marker, Flow Cytometry, Real-time Polymerase Chain Reaction

    11) Product Images from "Identification of Altered Developmental Pathways in Human Juvenile HD iPSC With 71Q and 109Q Using Transcriptome Profiling"

    Article Title: Identification of Altered Developmental Pathways in Human Juvenile HD iPSC With 71Q and 109Q Using Transcriptome Profiling

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00528

    qPCR validation of RNA-seq results. Seventeen genes in total were selected for DE confirmation in the same RNA samples used for RNA-seq. Among selected genes, 10 were DE in both HD iPSC lines (A) , 4 were DE only in HD71Q iPSC lines (B) , and 3 genes were DE only in HD109Q iPSC lines (C) , compared to control lines. Results for each analyzed gene are presented as a scatter plot of Log2 NRQ values for each sample ( n = 3 per genotype), together with Mean Log2 NRQ (thick horizontal line) for each genotype and 95% CIs (thin vertical lines). Dashed lines at 0 represent WT control lines. Genes are differentially expressed when 95% CI lines do not cross WT line. For easier reference, Log2 FC data from RNA-seq experiments (circles) were also included on plots. (D) Two additional reference genes, identified as not significantly dysregulated, were also selected for RNA-seq results validation.
    Figure Legend Snippet: qPCR validation of RNA-seq results. Seventeen genes in total were selected for DE confirmation in the same RNA samples used for RNA-seq. Among selected genes, 10 were DE in both HD iPSC lines (A) , 4 were DE only in HD71Q iPSC lines (B) , and 3 genes were DE only in HD109Q iPSC lines (C) , compared to control lines. Results for each analyzed gene are presented as a scatter plot of Log2 NRQ values for each sample ( n = 3 per genotype), together with Mean Log2 NRQ (thick horizontal line) for each genotype and 95% CIs (thin vertical lines). Dashed lines at 0 represent WT control lines. Genes are differentially expressed when 95% CI lines do not cross WT line. For easier reference, Log2 FC data from RNA-seq experiments (circles) were also included on plots. (D) Two additional reference genes, identified as not significantly dysregulated, were also selected for RNA-seq results validation.

    Techniques Used: Real-time Polymerase Chain Reaction, RNA Sequencing Assay

    12) Product Images from "Phosphorylation of CAD1, PLDdelta, NDT1, RPM1 Proteins Induce Resistance in Tomatoes Infected by Ralstonia solanacearum"

    Article Title: Phosphorylation of CAD1, PLDdelta, NDT1, RPM1 Proteins Induce Resistance in Tomatoes Infected by Ralstonia solanacearum

    Journal: Plants

    doi: 10.3390/plants11060726

    Time course of quantitative Real Time PCR analysis of four genes in susceptible and resistant tomato cultivars, Sidathip and Hawaii7996 inoculated with  Ralstonia solanacearum . Total RNA was extracted from the stems of resistant tomato cv. Hawaii7996 and susceptible tomato Sidathip at 15 min, 30 min, 24 h, and 48 h after inoculation with  Ralstonia solanacerum  or distilled water for control. The control samples collected from Hawaii7996 and Sidathip treated with water were used for calibration. The level of expression of the genes shows ( A ) phopholipase D delta, ( B ) RPM1, ( C ) cinnamyl alcohol dehydrogenase 1 (CAD1), and ( D ) nicotinamide adenine dinucleotide transporter 1 (NDT1). Error bar represents the standard deviation obtained from three biological replicates. Asterisks indicate the statistical significance level by  p -value obtained by two tailed student’s  t -test:  p -value
    Figure Legend Snippet: Time course of quantitative Real Time PCR analysis of four genes in susceptible and resistant tomato cultivars, Sidathip and Hawaii7996 inoculated with Ralstonia solanacearum . Total RNA was extracted from the stems of resistant tomato cv. Hawaii7996 and susceptible tomato Sidathip at 15 min, 30 min, 24 h, and 48 h after inoculation with Ralstonia solanacerum or distilled water for control. The control samples collected from Hawaii7996 and Sidathip treated with water were used for calibration. The level of expression of the genes shows ( A ) phopholipase D delta, ( B ) RPM1, ( C ) cinnamyl alcohol dehydrogenase 1 (CAD1), and ( D ) nicotinamide adenine dinucleotide transporter 1 (NDT1). Error bar represents the standard deviation obtained from three biological replicates. Asterisks indicate the statistical significance level by p -value obtained by two tailed student’s t -test: p -value

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

    13) Product Images from "Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy"

    Article Title: Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12880-9

    S100B’s ability to promote regeneration of acutely injured skeletal muscles requires RAGE at early, but not mid-late regeneration phase. ( a ) Injured  Ager −/−  TA muscles were injected with IgG or anti-S100B antibody at d4 p.i. and excised at d7 p.i. ( b ) Counts of interstitial cells and centrally nucleated myofibers (also see Fig.   S5h ). ( c ) PAX7 + , MyoD + , myogenin + , MAC3 +  and Ki67 +  cell counts (also see Fig.   S5i ). ( d ) Western blots of the indicated proteins in homogenates of IgG- and anti-S100B-treated  Ager –/–  muscles. Immunoblots of GAPDH and α-actinin are included as loading controls. Full-length blots are presented in Supplementary Fig.   S8  “Fig. 4”. ( e , f ) Macrophages isolated at d7 p.i. from injured  Ager −/−  muscles and analyzed by real-time PCR to measure the indicated macrophage markers ( e ) and  Tgfb  ( f ). Results are means ± SEM (n = 6). * p
    Figure Legend Snippet: S100B’s ability to promote regeneration of acutely injured skeletal muscles requires RAGE at early, but not mid-late regeneration phase. ( a ) Injured Ager −/− TA muscles were injected with IgG or anti-S100B antibody at d4 p.i. and excised at d7 p.i. ( b ) Counts of interstitial cells and centrally nucleated myofibers (also see Fig.  S5h ). ( c ) PAX7 + , MyoD + , myogenin + , MAC3 + and Ki67 + cell counts (also see Fig.  S5i ). ( d ) Western blots of the indicated proteins in homogenates of IgG- and anti-S100B-treated Ager –/– muscles. Immunoblots of GAPDH and α-actinin are included as loading controls. Full-length blots are presented in Supplementary Fig.  S8 “Fig. 4”. ( e , f ) Macrophages isolated at d7 p.i. from injured Ager −/− muscles and analyzed by real-time PCR to measure the indicated macrophage markers ( e ) and Tgfb ( f ). Results are means ± SEM (n = 6). * p

    Techniques Used: Injection, Western Blot, Isolation, Real-time Polymerase Chain Reaction

    S100B affects macrophages early after acute muscle injury. ( a ) TA muscles were treated as described in the legend to Fig.   1a . Muscles were excised at d3 or d7 p.i. ( b ) Counts of MAC3 +  cells. ( c – f,h ) Macrophages were isolated from IgG- and anti-S100B-treated injured muscles and either counted ( c ), analyzed by real-time PCR ( d , f , h ), or subjected to western blotting ( e ) (also see Fig.   S2c ). ( g ) Peritoneal macrophages were subjected to a migration assay using Boyden chambers in the presence of increasing S100B doses. ( i ) IgG- and anti-S100B-treated injured muscles excised at d7 p.i. and subjected to collagen IV detection by immunohistochemistry and western blotting. ( j ) Macrophages were isolated from IgG- and anti-S100B-treated injured muscles at d3 p.i., cultured for 24 h in the absence or presence of 200 ng S100B/ml, and analyzed by real-time PCR. ( k ) Macrophages were treated with IFN-γ, IL-10 or IL-4 in the absence or presence of the NF-κB inhibitor, Bay11-7085, and analyzed by real-time PCR for  S100b  levels. ( l ) Western blot analysis of S100B in conditioned media of IFN-γ-, IL-10- or IL-4-stimulated peritoneal macrophages. ( m ) Proliferation assay of C2C12 myoblasts cultured in the presence of IgG- or anti-S100B-treated conditioned media from vehicle- or IFN-γ-stimulated peritoneal macrophages (left) and differentiation assay of C2C12 myoblasts cultured in the presence of IgG- or anti-S100B-treated conditioned media from vehicle- or IL-10-stimulated peritoneal macrophages (right). Immunoblots of α-tubulin are included as loading controls in western blots in  e , m . Full-length blots are presented in Supplementary Fig.   S7  “Fig. 2”. Results are means ± SEM (n = 6).  * p
    Figure Legend Snippet: S100B affects macrophages early after acute muscle injury. ( a ) TA muscles were treated as described in the legend to Fig.  1a . Muscles were excised at d3 or d7 p.i. ( b ) Counts of MAC3 + cells. ( c – f,h ) Macrophages were isolated from IgG- and anti-S100B-treated injured muscles and either counted ( c ), analyzed by real-time PCR ( d , f , h ), or subjected to western blotting ( e ) (also see Fig.  S2c ). ( g ) Peritoneal macrophages were subjected to a migration assay using Boyden chambers in the presence of increasing S100B doses. ( i ) IgG- and anti-S100B-treated injured muscles excised at d7 p.i. and subjected to collagen IV detection by immunohistochemistry and western blotting. ( j ) Macrophages were isolated from IgG- and anti-S100B-treated injured muscles at d3 p.i., cultured for 24 h in the absence or presence of 200 ng S100B/ml, and analyzed by real-time PCR. ( k ) Macrophages were treated with IFN-γ, IL-10 or IL-4 in the absence or presence of the NF-κB inhibitor, Bay11-7085, and analyzed by real-time PCR for S100b levels. ( l ) Western blot analysis of S100B in conditioned media of IFN-γ-, IL-10- or IL-4-stimulated peritoneal macrophages. ( m ) Proliferation assay of C2C12 myoblasts cultured in the presence of IgG- or anti-S100B-treated conditioned media from vehicle- or IFN-γ-stimulated peritoneal macrophages (left) and differentiation assay of C2C12 myoblasts cultured in the presence of IgG- or anti-S100B-treated conditioned media from vehicle- or IL-10-stimulated peritoneal macrophages (right). Immunoblots of α-tubulin are included as loading controls in western blots in  e , m . Full-length blots are presented in Supplementary Fig.  S7 “Fig. 2”. Results are means ± SEM (n = 6). * p

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Western Blot, Migration, Immunohistochemistry, Cell Culture, Proliferation Assay, Differentiation Assay

    S100B is required during the macrophage M2 phase for efficient regeneration. ( a ) Injured TA muscles were injected with IgG or anti-S100B antibody at d4 p.i. Treated muscle were excised at d7 or d14 p.i. ( b ) Histology of muscle tissue (upper panel) and counts of interstitial cells and centrally nucleated myofibers (lower panel). ( c ) PAX7 + , MyoD + , myogenin + , MAC3 +  and Ki67 +  cell counts (also see Fig.   S4b ). ( d ) Western blots of the indicated proteins in homogenates of IgG- and anti-S100B-treated muscles. Immunoblots of GAPDH and α-actinin are included as loading controls. ( e ) Myofiber size distribution at d14 p.i. of uninjured muscles and IgG- and anti-S100B-treated injured muscles. ( f,g ) Macrophages isolated from IgG- and anti-S100B-treated injured muscles and analyzed by real-time PCR. Full-length blots are presented in Supplementary Fig.   S7  “Fig. 3”. Results are means ± SEM (n = 6). ** p
    Figure Legend Snippet: S100B is required during the macrophage M2 phase for efficient regeneration. ( a ) Injured TA muscles were injected with IgG or anti-S100B antibody at d4 p.i. Treated muscle were excised at d7 or d14 p.i. ( b ) Histology of muscle tissue (upper panel) and counts of interstitial cells and centrally nucleated myofibers (lower panel). ( c ) PAX7 + , MyoD + , myogenin + , MAC3 + and Ki67 + cell counts (also see Fig.  S4b ). ( d ) Western blots of the indicated proteins in homogenates of IgG- and anti-S100B-treated muscles. Immunoblots of GAPDH and α-actinin are included as loading controls. ( e ) Myofiber size distribution at d14 p.i. of uninjured muscles and IgG- and anti-S100B-treated injured muscles. ( f,g ) Macrophages isolated from IgG- and anti-S100B-treated injured muscles and analyzed by real-time PCR. Full-length blots are presented in Supplementary Fig.  S7 “Fig. 3”. Results are means ± SEM (n = 6). ** p

    Techniques Used: Injection, Western Blot, Isolation, Real-time Polymerase Chain Reaction

    Persistence of S100B at damage sites following acute muscle injury prolongs the M1 macrophage (inflammatory) phase and dampens muscle regeneration. ( a ) Injured wild-type or  Ager −/−  TA muscles were injected with vehicle or S100B at d1, d3 and d5 p.i., excised at d7 p.i. and analyzed as detailed in b-f for wild-type TA and in g for  Ager −/−  TA. ( b ) Histology and counts of interstitial cell and centrally nucleated myofiber numbers. ( c ) Muscle homogenates were subjected to western blotting for detection of the indicated proteins. Immunoblots of GAPDH and α-actinin are included as loading controls. Full-length blots are presented in Supplementary Fig.   S8  “Fig. 6”. ( d ) Counts of cell types based on immunohistochemistry for the indicated antigens. ( e ) Macrophages were isolated from muscles and subjected to real-time PCR for determination of levels of the indicated  genes. ( f ) Muscles were analyzed for MAC3 or collagen IV expression by immunohistochemistry and western blotting. Immunoblots of α-actinin are included as loading controls. Full-length blots are presented in Supplementary Fig.   S8  “Fig. 6”. ( g ) Injured  Ager −/−  TA muscles were injected with S100B as described in ( a ) and analyzed as described in ( b ). Results are means ± SEM (n = 6). ** p
    Figure Legend Snippet: Persistence of S100B at damage sites following acute muscle injury prolongs the M1 macrophage (inflammatory) phase and dampens muscle regeneration. ( a ) Injured wild-type or Ager −/− TA muscles were injected with vehicle or S100B at d1, d3 and d5 p.i., excised at d7 p.i. and analyzed as detailed in b-f for wild-type TA and in g for Ager −/− TA. ( b ) Histology and counts of interstitial cell and centrally nucleated myofiber numbers. ( c ) Muscle homogenates were subjected to western blotting for detection of the indicated proteins. Immunoblots of GAPDH and α-actinin are included as loading controls. Full-length blots are presented in Supplementary Fig.  S8 “Fig. 6”. ( d ) Counts of cell types based on immunohistochemistry for the indicated antigens. ( e ) Macrophages were isolated from muscles and subjected to real-time PCR for determination of levels of the indicated genes. ( f ) Muscles were analyzed for MAC3 or collagen IV expression by immunohistochemistry and western blotting. Immunoblots of α-actinin are included as loading controls. Full-length blots are presented in Supplementary Fig.  S8 “Fig. 6”. ( g ) Injured Ager −/− TA muscles were injected with S100B as described in ( a ) and analyzed as described in ( b ). Results are means ± SEM (n = 6). ** p

    Techniques Used: Injection, Western Blot, Immunohistochemistry, Isolation, Real-time Polymerase Chain Reaction, Expressing

    14) Product Images from "miR-7 Restores Phenotypes in Myotonic Dystrophy Muscle Cells by Repressing Hyperactivated Autophagy"

    Article Title: miR-7 Restores Phenotypes in Myotonic Dystrophy Muscle Cells by Repressing Hyperactivated Autophagy

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2019.11.012

    Impaired Expression of Autophagy-Related Genes Is Restored by Increasing miR-7 Levels in DM1 Myoblasts (A) Quantification of relative expression of autophagy-related genes ( ATG4A , ATG7 , ATG5 , ATG2B , ATG3 , and VPS34 ) in DM1 myoblasts by qRT-PCR using the 2 −ΔΔCt method. Green dashed line indicates the relative expression levels of the genes in CNT myoblasts. Logarithmic representation on base 2 (log 2 ) of the qRT-PCR quantification of (B) ATG4A , (C) ATG7 , (D) ATG5 , (E) ATG2B , (F) ATG3 , and (G) VPS34 in CNT (purple) and DM1 (blue) myoblasts transdifferentiated for 7 days after transfection with the indicated concentration of antagomiR-7 (in control TDMs) or agomiR-7 (in DM1 TDMs), respectively. Gene expression levels were normalized to cells transfected with antagomiR or agomiR scramble at each concentration. In all cases, GAPDH expression was used as reference gene (n = 3). Data were obtained using the 2 −ΔΔCt method. The bar graphs show mean ± SEM. *p
    Figure Legend Snippet: Impaired Expression of Autophagy-Related Genes Is Restored by Increasing miR-7 Levels in DM1 Myoblasts (A) Quantification of relative expression of autophagy-related genes ( ATG4A , ATG7 , ATG5 , ATG2B , ATG3 , and VPS34 ) in DM1 myoblasts by qRT-PCR using the 2 −ΔΔCt method. Green dashed line indicates the relative expression levels of the genes in CNT myoblasts. Logarithmic representation on base 2 (log 2 ) of the qRT-PCR quantification of (B) ATG4A , (C) ATG7 , (D) ATG5 , (E) ATG2B , (F) ATG3 , and (G) VPS34 in CNT (purple) and DM1 (blue) myoblasts transdifferentiated for 7 days after transfection with the indicated concentration of antagomiR-7 (in control TDMs) or agomiR-7 (in DM1 TDMs), respectively. Gene expression levels were normalized to cells transfected with antagomiR or agomiR scramble at each concentration. In all cases, GAPDH expression was used as reference gene (n = 3). Data were obtained using the 2 −ΔΔCt method. The bar graphs show mean ± SEM. *p

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

    15) Product Images from "CD32 Ligation Promotes the Activation of CD4+ T Cells"

    Article Title: CD32 Ligation Promotes the Activation of CD4+ T Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02814

    Activation of CD4+ T cells results in an increased expression of CD32. (A,B) PBMCs were cultured with medium (controls), IL-2 (20 ng/ml) or immobilized anti-CD3 (10 μg/ml) plus soluble anti-CD28 (1 mg/ml) (aCD3/aCD28) antibodies, during 18 or 36 h. The frequency of CD32+CD4+ T cells was analyzed by flow cytometry. (A) Representative dot plots of cell surface expression of CD32 in CD4+ T cells analyzed at 36 h of culture. Results are expressed as percentages on total lymphocytes. (B) Frequency of CD32+ cells on gated CD4+ T cells at 18 and 36 h of culture. (C–F) PBMCs were activated with either aCD3/aCD28 antibodies (C,D) or PHA (4 μg/ml, E,F ) for 36 h. Then, cell surface or intracellular expression of CD32 was analyzed. Cell surface expression of CD25 was also assessed. Cytoplasmic isotype control is shown in (C,E) . (G) Cell frequency of the different markers analyzed in CD32+CD4+ and CD32-CD4+ T cells. Results are expressed as percentage on each CD4+ T cell subset analyzed. (H) CD32a/CD32b mRNA ratio in activated CD32-CD4+ and CD32+CD4+ T cell subsets analyzed by qRT-PCR. Representative experiments are shown in (A,C,E) . Mean ± SEM of n donors are shown in (B) ( n = 7), (D) ( n = 7), (F) ( n = 7), (G) ( n = 8), and (H) ( n = 7). * p
    Figure Legend Snippet: Activation of CD4+ T cells results in an increased expression of CD32. (A,B) PBMCs were cultured with medium (controls), IL-2 (20 ng/ml) or immobilized anti-CD3 (10 μg/ml) plus soluble anti-CD28 (1 mg/ml) (aCD3/aCD28) antibodies, during 18 or 36 h. The frequency of CD32+CD4+ T cells was analyzed by flow cytometry. (A) Representative dot plots of cell surface expression of CD32 in CD4+ T cells analyzed at 36 h of culture. Results are expressed as percentages on total lymphocytes. (B) Frequency of CD32+ cells on gated CD4+ T cells at 18 and 36 h of culture. (C–F) PBMCs were activated with either aCD3/aCD28 antibodies (C,D) or PHA (4 μg/ml, E,F ) for 36 h. Then, cell surface or intracellular expression of CD32 was analyzed. Cell surface expression of CD25 was also assessed. Cytoplasmic isotype control is shown in (C,E) . (G) Cell frequency of the different markers analyzed in CD32+CD4+ and CD32-CD4+ T cells. Results are expressed as percentage on each CD4+ T cell subset analyzed. (H) CD32a/CD32b mRNA ratio in activated CD32-CD4+ and CD32+CD4+ T cell subsets analyzed by qRT-PCR. Representative experiments are shown in (A,C,E) . Mean ± SEM of n donors are shown in (B) ( n = 7), (D) ( n = 7), (F) ( n = 7), (G) ( n = 8), and (H) ( n = 7). * p

    Techniques Used: Activation Assay, Expressing, Cell Culture, Flow Cytometry, Quantitative RT-PCR

    16) Product Images from "Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages"

    Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03111

    Mechanisms of GILZ downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p
    Figure Legend Snippet: Mechanisms of GILZ downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p

    Techniques Used: Activation Assay, Affinity Magnetic Separation, Expressing, Western Blot, Quantitative RT-PCR

    17) Product Images from "A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2"

    Article Title: A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2

    Journal: medRxiv

    doi: 10.1101/2020.12.16.20246678

    Selection of a human housekeeping gene to be used as internal control. (A) Representative amplification curves for POLR2A and U1 using both the custom-made mix and the INBIO master mix. (B) Agarose gel electrophoresis of the products obtained after qPCR of 1:1, 1:16 dilutions and the negative control respectively, for each primer pair using both mixes.
    Figure Legend Snippet: Selection of a human housekeeping gene to be used as internal control. (A) Representative amplification curves for POLR2A and U1 using both the custom-made mix and the INBIO master mix. (B) Agarose gel electrophoresis of the products obtained after qPCR of 1:1, 1:16 dilutions and the negative control respectively, for each primer pair using both mixes.

    Techniques Used: Selection, Amplification, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Negative Control

    Using an intercalating dye can yield similar sensitivity and specificity as TaqMan probes. (A) Boxplot of the Ct shift for the SYBR Green RT-qPCR (RdRP amplicon) versus two different TaqMan-based reactions (N and ORF1ab amplicons) using the Charité protocol. (B) RT-qPCR amplification curves for serial dilutions of a SARS-CoV-2 standard RNA, from 4×10 5 copies per µL (c/µL) to 4 c/µL, using either the unmodified Charité protocol (TaqMan) or the one adapted for an intercalating dye (SYBR Green). (C) Summary of the performance obtained using the different commercial and customized RT-qPCR protocols evaluated. (D) Ct values for each sample (arbitrarily numbered) tested with every RT-qPCR protocol developed and optimized in this work. Not all samples were evaluated with all protocols. Missing points represent negative (not amplified) samples. (E) A drop in diagnostic sensitivity using two-step RT-qPCR approaches is circumscribed to samples with higher Ct values. Table summarizing the sensitivity observed using either the INBIO master mix or the custom-made mix when separating the positive samples in terciles (T1-T3) according to the previously assessed Ct value using the RT-qPCR DisCoVery kit (ORF1ab and N amplicons). (F) RT-qPCR amplification curves for either serial dilutions of a pool of positive samples ( top panel ) or random samples ( bottom panel ), using four different retro-transcriptases for cDNA synthesis.
    Figure Legend Snippet: Using an intercalating dye can yield similar sensitivity and specificity as TaqMan probes. (A) Boxplot of the Ct shift for the SYBR Green RT-qPCR (RdRP amplicon) versus two different TaqMan-based reactions (N and ORF1ab amplicons) using the Charité protocol. (B) RT-qPCR amplification curves for serial dilutions of a SARS-CoV-2 standard RNA, from 4×10 5 copies per µL (c/µL) to 4 c/µL, using either the unmodified Charité protocol (TaqMan) or the one adapted for an intercalating dye (SYBR Green). (C) Summary of the performance obtained using the different commercial and customized RT-qPCR protocols evaluated. (D) Ct values for each sample (arbitrarily numbered) tested with every RT-qPCR protocol developed and optimized in this work. Not all samples were evaluated with all protocols. Missing points represent negative (not amplified) samples. (E) A drop in diagnostic sensitivity using two-step RT-qPCR approaches is circumscribed to samples with higher Ct values. Table summarizing the sensitivity observed using either the INBIO master mix or the custom-made mix when separating the positive samples in terciles (T1-T3) according to the previously assessed Ct value using the RT-qPCR DisCoVery kit (ORF1ab and N amplicons). (F) RT-qPCR amplification curves for either serial dilutions of a pool of positive samples ( top panel ) or random samples ( bottom panel ), using four different retro-transcriptases for cDNA synthesis.

    Techniques Used: SYBR Green Assay, Quantitative RT-PCR, Amplification, Diagnostic Assay

    18) Product Images from "Trypanosoma cruzi 80 kDa prolyl oligopeptidase (Tc80) as a novel immunogen for Chagas disease vaccine"

    Article Title: Trypanosoma cruzi 80 kDa prolyl oligopeptidase (Tc80) as a novel immunogen for Chagas disease vaccine

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006384

    Protection in a chronic infection model in vaccinated mice. Animals were immunized as indicated and challenged 15 days later with T . cruzi K98 strain. a) Parasitemia curve (I) and its AUC (II) during the acute phase of infection. b) Serum level of tissue damage-associated enzymes at 100 dpi: creatine kinase, CK (I) and its cardiac isoform, CK-MB(II); glutamate oxaloacetate transaminase, GOT (III); and lactate dehydrogenase, LDH (IV). c) Electrocardiogram parameters at 100 dpi: Corrected QT interval(cQTi) (I) and PR interval(PRi) (II). d) Parasite load by qPCR in target tissues at 100 dpi. e) Histopathological analysis of skeletal and heart muscle at 100 dpi. Representative muscle sections stained with hematoxilin-eosin at 100x magnification (I) and semi-quantitative analysis of inflammatory infiltrate (II). Results are expressed as mean ± SEM (n = 4–5 per group) and are representative of two independent experiments. Survival statistical analysis was performed with log-rank test. *p
    Figure Legend Snippet: Protection in a chronic infection model in vaccinated mice. Animals were immunized as indicated and challenged 15 days later with T . cruzi K98 strain. a) Parasitemia curve (I) and its AUC (II) during the acute phase of infection. b) Serum level of tissue damage-associated enzymes at 100 dpi: creatine kinase, CK (I) and its cardiac isoform, CK-MB(II); glutamate oxaloacetate transaminase, GOT (III); and lactate dehydrogenase, LDH (IV). c) Electrocardiogram parameters at 100 dpi: Corrected QT interval(cQTi) (I) and PR interval(PRi) (II). d) Parasite load by qPCR in target tissues at 100 dpi. e) Histopathological analysis of skeletal and heart muscle at 100 dpi. Representative muscle sections stained with hematoxilin-eosin at 100x magnification (I) and semi-quantitative analysis of inflammatory infiltrate (II). Results are expressed as mean ± SEM (n = 4–5 per group) and are representative of two independent experiments. Survival statistical analysis was performed with log-rank test. *p

    Techniques Used: Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Staining

    19) Product Images from "Azelastine potentiates antiasthmatic dexamethasone effect on a murine asthma model, et al. Azelastine potentiates antiasthmatic dexamethasone effect on a murine asthma model"

    Article Title: Azelastine potentiates antiasthmatic dexamethasone effect on a murine asthma model, et al. Azelastine potentiates antiasthmatic dexamethasone effect on a murine asthma model

    Journal: Pharmacology Research & Perspectives

    doi: 10.1002/prp2.531

    Effect of cotreatment on asthma‐inflammatory genes expression. Transcriptional response of four asthma‐inflammatory genes (A) IL‐4, (B) IL‐5, (C) Muc5AC, and (D) ArgI from lungs of all experimental mice. mRNA levels were quantified by qPCR as described in the methods section. N: Naïve animals; OVA: animals sensitized and treated with vehicle; AZE: animals sensitized with OVA and intranasally treated with azelastine; SD: animals sensitized with OVA and intranasally treated with a 10‐fold dilution of optimal dexamethasone solution. OD: animals sensitized with OVA and intranasally treated with an optimal dose of dexamethasone. AD: animals sensitized with OVA and intranasally treated with a solution containing azelastine and a suboptimal dose of dexamethasone. Results are mean ± SD. * P
    Figure Legend Snippet: Effect of cotreatment on asthma‐inflammatory genes expression. Transcriptional response of four asthma‐inflammatory genes (A) IL‐4, (B) IL‐5, (C) Muc5AC, and (D) ArgI from lungs of all experimental mice. mRNA levels were quantified by qPCR as described in the methods section. N: Naïve animals; OVA: animals sensitized and treated with vehicle; AZE: animals sensitized with OVA and intranasally treated with azelastine; SD: animals sensitized with OVA and intranasally treated with a 10‐fold dilution of optimal dexamethasone solution. OD: animals sensitized with OVA and intranasally treated with an optimal dose of dexamethasone. AD: animals sensitized with OVA and intranasally treated with a solution containing azelastine and a suboptimal dose of dexamethasone. Results are mean ± SD. * P

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

    20) Product Images from "2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study"

    Article Title: 2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0221942

    Induction of pro inflammatory factors by chronic H 2 O 2 treatment. Quantitative PCR gene expression analysis of 2D- and 3D-HTMC subjected 500μM for 48 h and 72 h. IL-1α IL-1β, and IL-6 (Panels A, B and C, respectively). Data are expressed as fold-increase relative to the 2D control at the same end-point and normalized to Ubiquitin housekeeping gene expression. Each bar represents the mean ± S.D. of three independent experiments performed in triplicate. (Panel D) The figures depicted are representative of at least three similar immunoblot analysis of NF-kB (p65), p-NF-kB (p65) protein levels in untreated HTMCs and treated HTMCs (H 2 O 2 ) whole protein lysates at indicated time points. GAPDH was used as an internal control for equal protein loading on the gel. (Panel E) NF-kBp65 activation was evaluated in HTMC cells subjected to chronic treatment with H 2 O 2 for 24, 48 and 72 hrs. The analysis was performed by immunoblotting and the bars represent the ratio of phosfoNF-kBp65/NF-kBp65, and are expressed as fold vs. untreated HTMC cultures. Data represent the mean ± S.D. of 3 independent experiments. ***p
    Figure Legend Snippet: Induction of pro inflammatory factors by chronic H 2 O 2 treatment. Quantitative PCR gene expression analysis of 2D- and 3D-HTMC subjected 500μM for 48 h and 72 h. IL-1α IL-1β, and IL-6 (Panels A, B and C, respectively). Data are expressed as fold-increase relative to the 2D control at the same end-point and normalized to Ubiquitin housekeeping gene expression. Each bar represents the mean ± S.D. of three independent experiments performed in triplicate. (Panel D) The figures depicted are representative of at least three similar immunoblot analysis of NF-kB (p65), p-NF-kB (p65) protein levels in untreated HTMCs and treated HTMCs (H 2 O 2 ) whole protein lysates at indicated time points. GAPDH was used as an internal control for equal protein loading on the gel. (Panel E) NF-kBp65 activation was evaluated in HTMC cells subjected to chronic treatment with H 2 O 2 for 24, 48 and 72 hrs. The analysis was performed by immunoblotting and the bars represent the ratio of phosfoNF-kBp65/NF-kBp65, and are expressed as fold vs. untreated HTMC cultures. Data represent the mean ± S.D. of 3 independent experiments. ***p

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

    21) Product Images from "Serum from Older Adults Increases Apoptosis and Molecular Aging Markers in Human Hippocampal Progenitor Cells"

    Article Title: Serum from Older Adults Increases Apoptosis and Molecular Aging Markers in Human Hippocampal Progenitor Cells

    Journal: Aging and Disease

    doi: 10.14336/AD.2021.0409

    Validation of differentially expressed genes in response to serum from older adults. (A, B) Networks formed by differentially expressed genes in response to young and old serum. 111 differentially expressed genes during differentiation of hippocampal progenitors in the presence of young or old serum were analysed using Ingenuity Pathway Analysis (IPA) to assess network relationships between candidate genes, A) top network associated functions included “System Development and Function” and “Cell to Cell Signalling”, B) second highest network associated functions included “Cellular Development”, “Cellular Growth and Proliferation” and “Cancer”. (A, B) significance of genes reflected on continuous colour scale from red (most significant) to light pink (least significant). Inferred molecular interactions identified by IPA are shown in grey. (C-F) Validation by qPCR of differentially expressed microarray candidate genes in response to young or old serum. Relative expression of microarray candidates C) transmembrane 149 ( TMEM149 ) D) ring finger protein 126 (RNF126) E) mitogen-activated protein kinase 7 ( MAP3K7 ) F) endonuclease G ( ENDOG ) normalised to one young-serum induced readout, corresponding to 1 on the y axis for both the microarray and qPCR validation. Each green circle (young, n = 3) or red square (old, n = 3) represents expression values during differentiation of hippocampal progenitors in the presence of human serum at the stated time point. Microarray statistics not included. Unpaired student one-tailed t-test conducted on qPCR data, * P
    Figure Legend Snippet: Validation of differentially expressed genes in response to serum from older adults. (A, B) Networks formed by differentially expressed genes in response to young and old serum. 111 differentially expressed genes during differentiation of hippocampal progenitors in the presence of young or old serum were analysed using Ingenuity Pathway Analysis (IPA) to assess network relationships between candidate genes, A) top network associated functions included “System Development and Function” and “Cell to Cell Signalling”, B) second highest network associated functions included “Cellular Development”, “Cellular Growth and Proliferation” and “Cancer”. (A, B) significance of genes reflected on continuous colour scale from red (most significant) to light pink (least significant). Inferred molecular interactions identified by IPA are shown in grey. (C-F) Validation by qPCR of differentially expressed microarray candidate genes in response to young or old serum. Relative expression of microarray candidates C) transmembrane 149 ( TMEM149 ) D) ring finger protein 126 (RNF126) E) mitogen-activated protein kinase 7 ( MAP3K7 ) F) endonuclease G ( ENDOG ) normalised to one young-serum induced readout, corresponding to 1 on the y axis for both the microarray and qPCR validation. Each green circle (young, n = 3) or red square (old, n = 3) represents expression values during differentiation of hippocampal progenitors in the presence of human serum at the stated time point. Microarray statistics not included. Unpaired student one-tailed t-test conducted on qPCR data, * P

    Techniques Used: Indirect Immunoperoxidase Assay, Real-time Polymerase Chain Reaction, Microarray, Expressing, One-tailed Test

    Expression of genes involved in maintaining genomic integrity and proteostasis following culture with young or old serum at 6 hours and 6 days differentiation. Relative expression of (A, B) Poly ADP-ribose polymerase 1 ( PARP1 ), (C, D) Telomerase reverse transcriptase ( TERT) and (E, F) Ubiquitin carboxyl-terminal esterase L1 (UCHL1), normalised to one young subject (21 years), corresponding to 1 on the y axis. Each green circle (young serum, n = 17, mean age of 25.6 years) or red square (old serum, n = 23, mean age of 78 years) represents n =2 technical replicates following analysis of qPCR data, after 6 hrs (hours) or 6 days (d) differentiation of hippocampal progenitors in presence of human serum. Unpaired two-tailed student and Mann-Whitney t-tests as appropriate, ** P
    Figure Legend Snippet: Expression of genes involved in maintaining genomic integrity and proteostasis following culture with young or old serum at 6 hours and 6 days differentiation. Relative expression of (A, B) Poly ADP-ribose polymerase 1 ( PARP1 ), (C, D) Telomerase reverse transcriptase ( TERT) and (E, F) Ubiquitin carboxyl-terminal esterase L1 (UCHL1), normalised to one young subject (21 years), corresponding to 1 on the y axis. Each green circle (young serum, n = 17, mean age of 25.6 years) or red square (old serum, n = 23, mean age of 78 years) represents n =2 technical replicates following analysis of qPCR data, after 6 hrs (hours) or 6 days (d) differentiation of hippocampal progenitors in presence of human serum. Unpaired two-tailed student and Mann-Whitney t-tests as appropriate, ** P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

    Serum assay timeline and optimal serum concentration. (A) Optimised assay timeline to evaluate the impacts of the human systemic environment on human. (B) Experimental timeline for qPCR and whole-genome expression analysis by microarray. (C-G) Human serum concentration dose-response curves for the differentiation phase of the assay. Culturing human hippocampal progenitors with increasing concentrations of serum (as a percentage on the x axis) increased (C) cell number, reduced (D) apoptotic cell death (CC3 as % of total cells) (E) immature neurons as measured (Map2 as % of total cells), and increased (F) astrogliogenesis (S100β as % of total cells). (G) Representative images of a dose-dependent decrease in Map2 staining (red fluorescence), also highlighting the increase in cell number through DAPI staining of cell nuclei. n = 3, using serum from a young male (aged 23 years). one-way ANOVA conducted across all concentrations (post-hoc comparisons not presented), ** P
    Figure Legend Snippet: Serum assay timeline and optimal serum concentration. (A) Optimised assay timeline to evaluate the impacts of the human systemic environment on human. (B) Experimental timeline for qPCR and whole-genome expression analysis by microarray. (C-G) Human serum concentration dose-response curves for the differentiation phase of the assay. Culturing human hippocampal progenitors with increasing concentrations of serum (as a percentage on the x axis) increased (C) cell number, reduced (D) apoptotic cell death (CC3 as % of total cells) (E) immature neurons as measured (Map2 as % of total cells), and increased (F) astrogliogenesis (S100β as % of total cells). (G) Representative images of a dose-dependent decrease in Map2 staining (red fluorescence), also highlighting the increase in cell number through DAPI staining of cell nuclei. n = 3, using serum from a young male (aged 23 years). one-way ANOVA conducted across all concentrations (post-hoc comparisons not presented), ** P

    Techniques Used: Serum Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing, Microarray, Staining, Fluorescence

    22) Product Images from "A novel Axin2 knock‐in mouse model for visualization and lineage tracing of WNT/ CTNNB1 responsive cells. A novel Axin2 knock‐in mouse model for visualization and lineage tracing of WNT/ CTNNB1 responsive cells"

    Article Title: A novel Axin2 knock‐in mouse model for visualization and lineage tracing of WNT/ CTNNB1 responsive cells. A novel Axin2 knock‐in mouse model for visualization and lineage tracing of WNT/ CTNNB1 responsive cells

    Journal: Genesis (New York, N.y. : 2000)

    doi: 10.1002/dvg.23387

    Design and generation of the Axin2 P2A‐rtTA3‐T2A‐3xNLS‐SGFP2 knock‐in allele. (a) Overview of WNT/CTNNB1 reporter and lineage tracing strains that are available from public repositories. Information was retrieved from the International Mouse Strain Resource (IMSR) at http://www.findmice.org . Strains are subdivided by four criteria: whether it is a random transgenic insertion (Tg) or a targeted insertion ( Rosa26 or Axin2 locus); whether the targeted Axin2 allele still produces a functional AXIN2 protein; whether the strain carries a reporter that can directly visualize WNT/CTNNB1 responsive cells; whether the strain can be used for lineage tracing experiments. n.a., not applicable, FP, fluorescent protein, asterisk “*” requires Cre‐mediated removal of a stop‐cassette before the reporter becomes functional. (b) Schematic representation of the mouse Axin2 locus (chr11:108920349‐108950781, mm10 coordinates). Most existing Axin2 knock‐in strains target the 5′ end of the gene by introducing the knock‐in cassette at the start codon (ATG) in exon 2. This disrupts the endogenous Axin2 coding sequence. (c) Cartoon depicting the Axin2 P2A‐rtTA3‐T2A‐3xNLS‐SGFP2 targeting construct. A multi‐cistronic targeting cassette was cloned immediately upstream of the Axin2 stop codon (TGA) in exon 11. The PGK‐Neo‐polyA cassette, flanked by FRT sites (indicated by triangles), was used for selection of embryonic stem cells and was removed prior to establishing the colony. (d) Targeted locus after removal of the PGK‐Neo‐polyA selection cassette. The approximate location of the primers used for the genotyping PCR depicted in (g) is indicated with equilibrium arrows. (e) The 3′ knock‐in allele is designed to give rise to a single transcript in which the Axin2 5′ UTR, coding sequence and 3′ UTR are left intact. (f) Following translation, the self‐cleaving P2A and T2A sequences ensure that the polypeptide is cleaved into a fully functional AXIN2 protein, a doxycycline activatable rtTA3 driver for lineage tracing, and a bright green fluorescent protein that localizes to the nucleus (3xNLS‐SGFP2). (g) Genotyping PCR of wildtype, heterozygous and homozygous animals. Wildtype allele = 257 bp, knock‐in allele = 461 bp
    Figure Legend Snippet: Design and generation of the Axin2 P2A‐rtTA3‐T2A‐3xNLS‐SGFP2 knock‐in allele. (a) Overview of WNT/CTNNB1 reporter and lineage tracing strains that are available from public repositories. Information was retrieved from the International Mouse Strain Resource (IMSR) at http://www.findmice.org . Strains are subdivided by four criteria: whether it is a random transgenic insertion (Tg) or a targeted insertion ( Rosa26 or Axin2 locus); whether the targeted Axin2 allele still produces a functional AXIN2 protein; whether the strain carries a reporter that can directly visualize WNT/CTNNB1 responsive cells; whether the strain can be used for lineage tracing experiments. n.a., not applicable, FP, fluorescent protein, asterisk “*” requires Cre‐mediated removal of a stop‐cassette before the reporter becomes functional. (b) Schematic representation of the mouse Axin2 locus (chr11:108920349‐108950781, mm10 coordinates). Most existing Axin2 knock‐in strains target the 5′ end of the gene by introducing the knock‐in cassette at the start codon (ATG) in exon 2. This disrupts the endogenous Axin2 coding sequence. (c) Cartoon depicting the Axin2 P2A‐rtTA3‐T2A‐3xNLS‐SGFP2 targeting construct. A multi‐cistronic targeting cassette was cloned immediately upstream of the Axin2 stop codon (TGA) in exon 11. The PGK‐Neo‐polyA cassette, flanked by FRT sites (indicated by triangles), was used for selection of embryonic stem cells and was removed prior to establishing the colony. (d) Targeted locus after removal of the PGK‐Neo‐polyA selection cassette. The approximate location of the primers used for the genotyping PCR depicted in (g) is indicated with equilibrium arrows. (e) The 3′ knock‐in allele is designed to give rise to a single transcript in which the Axin2 5′ UTR, coding sequence and 3′ UTR are left intact. (f) Following translation, the self‐cleaving P2A and T2A sequences ensure that the polypeptide is cleaved into a fully functional AXIN2 protein, a doxycycline activatable rtTA3 driver for lineage tracing, and a bright green fluorescent protein that localizes to the nucleus (3xNLS‐SGFP2). (g) Genotyping PCR of wildtype, heterozygous and homozygous animals. Wildtype allele = 257 bp, knock‐in allele = 461 bp

    Techniques Used: Knock-In, Transgenic Assay, Functional Assay, Sequencing, Construct, Clone Assay, Selection, Genotyping Assay, Polymerase Chain Reaction

    Functional validation of the Axin2 P2A‐rtTA3‐T2A‐3xNLS‐SGFP2 allele. (a and b) Cartoons depicting the response of the knock‐in allele to downstream WNT/CTNNB1 signaling, either by activation by WNT proteins in vivo (a) or by treatment with CHIR99021, a specific GSK3 inhibitor (b). Cells with active WNT/CTNNB1 signaling will induce Axin2 , resulting in the expression of 3xNLS‐sGFP expression (depicted as green nuclei). (c–f) Dot plots showing the dose‐dependent induction of Axin2 mRNA expression in wildtype (c) and heterozygous (d) mouse embryonic fibroblasts (MEFs), as measured by qRT‐PCR in n = 3 independent MEF isolates. Rpl13a was used as a reference gene, values in the DMSO treated control were set to 1. (e and f) Same as for (c and d), but showing rtTA3 (e) and SGFP2 (f) mRNA expression in heterozygous MEFs. (g) Confocal microscopy images of fixed mouse embryonic fibroblasts (MEFs), showing the dose‐dependent induction and direct detection of SGFP2. Scalebar is 100 μm. (h) Quantification of the experiment described and depicted in (g). Fixed MEFs were counterstained with DAPI to allow nuclear segmentation, after which the relative SGFP2 expression levels were calculated by correcting for the fluorescence intensity of the DAPI signal. This experiment was performed for n = 3 independent MEF isolates. One experiment is shown here. The results for two additional MEF isolates are shown in Figure S2 . (i) The rtTA3 transcriptional activator is induced together with SGFP2, but only becomes active in the presence of doxycycline (DOX). This results in activation of tetO ‐driven transgenes ( tetO‐Tg ). (j) Western blot showing the WNT/CTNNB1‐dependent induction of SGFP2 and the WNT/CTNNB1‐ and DOX‐dependent induction of a FLAG‐tagged protein expressed from a tetO ‐responsive allele in whole cell lysates from MEFs after treatment with different combinations of CHIR99021 (5 μM) and DOX (1 μg/ml). These MEFs were isolated from embryos carrying both the Axin2 P2A‐rtTA3‐T2A‐3xNLS‐SGFP2 knock‐in allele and a tetO ‐responsive FLAG‐Wnt5a transgene
    Figure Legend Snippet: Functional validation of the Axin2 P2A‐rtTA3‐T2A‐3xNLS‐SGFP2 allele. (a and b) Cartoons depicting the response of the knock‐in allele to downstream WNT/CTNNB1 signaling, either by activation by WNT proteins in vivo (a) or by treatment with CHIR99021, a specific GSK3 inhibitor (b). Cells with active WNT/CTNNB1 signaling will induce Axin2 , resulting in the expression of 3xNLS‐sGFP expression (depicted as green nuclei). (c–f) Dot plots showing the dose‐dependent induction of Axin2 mRNA expression in wildtype (c) and heterozygous (d) mouse embryonic fibroblasts (MEFs), as measured by qRT‐PCR in n = 3 independent MEF isolates. Rpl13a was used as a reference gene, values in the DMSO treated control were set to 1. (e and f) Same as for (c and d), but showing rtTA3 (e) and SGFP2 (f) mRNA expression in heterozygous MEFs. (g) Confocal microscopy images of fixed mouse embryonic fibroblasts (MEFs), showing the dose‐dependent induction and direct detection of SGFP2. Scalebar is 100 μm. (h) Quantification of the experiment described and depicted in (g). Fixed MEFs were counterstained with DAPI to allow nuclear segmentation, after which the relative SGFP2 expression levels were calculated by correcting for the fluorescence intensity of the DAPI signal. This experiment was performed for n = 3 independent MEF isolates. One experiment is shown here. The results for two additional MEF isolates are shown in Figure S2 . (i) The rtTA3 transcriptional activator is induced together with SGFP2, but only becomes active in the presence of doxycycline (DOX). This results in activation of tetO ‐driven transgenes ( tetO‐Tg ). (j) Western blot showing the WNT/CTNNB1‐dependent induction of SGFP2 and the WNT/CTNNB1‐ and DOX‐dependent induction of a FLAG‐tagged protein expressed from a tetO ‐responsive allele in whole cell lysates from MEFs after treatment with different combinations of CHIR99021 (5 μM) and DOX (1 μg/ml). These MEFs were isolated from embryos carrying both the Axin2 P2A‐rtTA3‐T2A‐3xNLS‐SGFP2 knock‐in allele and a tetO ‐responsive FLAG‐Wnt5a transgene

    Techniques Used: Functional Assay, Knock-In, Activation Assay, In Vivo, Expressing, Quantitative RT-PCR, Confocal Microscopy, Fluorescence, Western Blot, Isolation

    23) Product Images from "A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2"

    Article Title: A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2

    Journal: RNA Biology

    doi: 10.1080/15476286.2021.1926648

    Using an intercalating dye can yield similar sensitivity and specificity as TaqMan probes. (A) Paired boxplot of the Ct shift for the SYBR Green RT-qPCR (RdRP amplicon) versus two different TaqMan-based reactions (N and ORF1ab amplicons) using the Charité protocol. (B) RT-qPCR amplification curves for serial dilutions of a SARS-CoV-2 standard RNA, from 4 × 10 5 copies per µL (c/µL) to 4 c/µL, using either the unmodified Charité protocol (TaqMan) or the one adapted for an intercalating dye (SYBR Green). (C) Summary of the performance obtained using the different commercial and customized RT-qPCR protocols evaluated. (D) Ct values for each sample tested with every RT-qPCR protocol developed and optimized in this work. Not all samples were evaluated with all protocols. The cut-off for positivity for each mix is shown with an orange line. Diagnostic was done with a TaqMan commercial kit. Negative samples with undetermined Ct were assigned a pseudo -Ct of 41. (E) A drop in diagnostic sensitivity using two-step RT-qPCR approaches is circumscribed to samples with higher Ct values. Table summarizing the sensitivity observed using either the INBIO master mix or the custom-made mix when separating the positive samples in terciles (T1-T3) according to the previously assessed Ct value using the RT-qPCR DisCoVery kit (ORF1ab and N amplicons). (F) RT-qPCR amplification curves for either serial dilutions of a pool of positive samples ( top panel ) or random samples ( bottom panel ), using four different retro-transcriptases for cDNA synthesis
    Figure Legend Snippet: Using an intercalating dye can yield similar sensitivity and specificity as TaqMan probes. (A) Paired boxplot of the Ct shift for the SYBR Green RT-qPCR (RdRP amplicon) versus two different TaqMan-based reactions (N and ORF1ab amplicons) using the Charité protocol. (B) RT-qPCR amplification curves for serial dilutions of a SARS-CoV-2 standard RNA, from 4 × 10 5 copies per µL (c/µL) to 4 c/µL, using either the unmodified Charité protocol (TaqMan) or the one adapted for an intercalating dye (SYBR Green). (C) Summary of the performance obtained using the different commercial and customized RT-qPCR protocols evaluated. (D) Ct values for each sample tested with every RT-qPCR protocol developed and optimized in this work. Not all samples were evaluated with all protocols. The cut-off for positivity for each mix is shown with an orange line. Diagnostic was done with a TaqMan commercial kit. Negative samples with undetermined Ct were assigned a pseudo -Ct of 41. (E) A drop in diagnostic sensitivity using two-step RT-qPCR approaches is circumscribed to samples with higher Ct values. Table summarizing the sensitivity observed using either the INBIO master mix or the custom-made mix when separating the positive samples in terciles (T1-T3) according to the previously assessed Ct value using the RT-qPCR DisCoVery kit (ORF1ab and N amplicons). (F) RT-qPCR amplification curves for either serial dilutions of a pool of positive samples ( top panel ) or random samples ( bottom panel ), using four different retro-transcriptases for cDNA synthesis

    Techniques Used: SYBR Green Assay, Quantitative RT-PCR, Amplification, Diagnostic Assay

    Selection of a human housekeeping gene to be used as internal control. (A) Representative amplification curves for POLR2A and U1 using both the custom-made mix and the INBIO master mix. (B) Agarose gel electrophoresis of the products obtained after qPCR of 1:1, 1:16 dilutions and the negative control respectively, for each primer pair using both mixes
    Figure Legend Snippet: Selection of a human housekeeping gene to be used as internal control. (A) Representative amplification curves for POLR2A and U1 using both the custom-made mix and the INBIO master mix. (B) Agarose gel electrophoresis of the products obtained after qPCR of 1:1, 1:16 dilutions and the negative control respectively, for each primer pair using both mixes

    Techniques Used: Selection, Amplification, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Negative Control

    24) Product Images from "Cell type and tissue specific function of islet genes in zebrafish pancreas development"

    Article Title: Cell type and tissue specific function of islet genes in zebrafish pancreas development

    Journal: Developmental Biology

    doi: 10.1016/j.ydbio.2013.03.009

    Overlapping activities of three isl -genes in exocrine tissue formation. (A, B) Pancreatic ptf1 / p48 mRNA expression is similar in control (A) and isl1 −/− +MO isl2a/2b embryos (B) at 48 hpf. (C, D) Differently, pancreatic mnr2a mRNA expression is reduced in isl1 −/− +MO isl2a/2b embryos as compared to control embryos. (E, G) pdx1 mRNA expression at 80 hpf reveals a similar morphology of the extrapancreatic duct (arrow) in control and isl1 −/− +MO isl2a/2b embryos. Note the reduced gut specific pdx1 signals (asterisk) in the isl -depleted embryos. (G, H) trypsin mRNA expressing in exocrine tissue of a control embryo (G) and in an isl1 −/− +MO isl2a/2b embryos (G) at 76 hpf. (I) Quantitative analyses on the length of trypsin mRNA expression domain in control and triple isl gene deficient embryos at 76 hpf. Embryos are shown from ventral with anterior to the left (A–F) or dorsal with anterior to the left (G, H). P -values for significant changes are indicated. (J) qPCR analysis for mRNA expression levels of trypsin and ela3l in 96 hpf old whole embryos, normalized to EF1α . Coloring of the bars corresponds to the same genotypes presented in (I). Bars show mean+SEM.
    Figure Legend Snippet: Overlapping activities of three isl -genes in exocrine tissue formation. (A, B) Pancreatic ptf1 / p48 mRNA expression is similar in control (A) and isl1 −/− +MO isl2a/2b embryos (B) at 48 hpf. (C, D) Differently, pancreatic mnr2a mRNA expression is reduced in isl1 −/− +MO isl2a/2b embryos as compared to control embryos. (E, G) pdx1 mRNA expression at 80 hpf reveals a similar morphology of the extrapancreatic duct (arrow) in control and isl1 −/− +MO isl2a/2b embryos. Note the reduced gut specific pdx1 signals (asterisk) in the isl -depleted embryos. (G, H) trypsin mRNA expressing in exocrine tissue of a control embryo (G) and in an isl1 −/− +MO isl2a/2b embryos (G) at 76 hpf. (I) Quantitative analyses on the length of trypsin mRNA expression domain in control and triple isl gene deficient embryos at 76 hpf. Embryos are shown from ventral with anterior to the left (A–F) or dorsal with anterior to the left (G, H). P -values for significant changes are indicated. (J) qPCR analysis for mRNA expression levels of trypsin and ela3l in 96 hpf old whole embryos, normalized to EF1α . Coloring of the bars corresponds to the same genotypes presented in (I). Bars show mean+SEM.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Reduced expression of endocrine hormones in isl1 mutants. (A–F) Confocal image projections of the pancreatic islet in control (A–C) and isl1 −/− embryos (D–F) that were co-immunostained for Ins (green) and Sst (red) at 28 hpf (A, D), 48 hpf (B, E) and 76 hpf (C, F). (G–L) Immunostainings for Gcg (purple) in control (G–I) and in isl1 −/− embryos (J–L) at 48 hpf (G, J), 76 hpf (H, K) and 96 hpf (I, L). All embryos are shown from ventral with the anterior to the left. (M) Quantitative analysis of hormone expressing cells in control and isl1 −/− embryos. Bars show mean+SEM. (N) Relative expression levels of ins , gcga and sst2 mRNA in isl1 −/− mutant fish in relation to control littermates as revealed by qPCR analyses of whole embryo RNA preparations, normalized to EF1α .
    Figure Legend Snippet: Reduced expression of endocrine hormones in isl1 mutants. (A–F) Confocal image projections of the pancreatic islet in control (A–C) and isl1 −/− embryos (D–F) that were co-immunostained for Ins (green) and Sst (red) at 28 hpf (A, D), 48 hpf (B, E) and 76 hpf (C, F). (G–L) Immunostainings for Gcg (purple) in control (G–I) and in isl1 −/− embryos (J–L) at 48 hpf (G, J), 76 hpf (H, K) and 96 hpf (I, L). All embryos are shown from ventral with the anterior to the left. (M) Quantitative analysis of hormone expressing cells in control and isl1 −/− embryos. Bars show mean+SEM. (N) Relative expression levels of ins , gcga and sst2 mRNA in isl1 −/− mutant fish in relation to control littermates as revealed by qPCR analyses of whole embryo RNA preparations, normalized to EF1α .

    Techniques Used: Expressing, Mutagenesis, Fluorescence In Situ Hybridization, Real-time Polymerase Chain Reaction

    Unchanged expression of early endocrine markers in isl1 mutants. Whole mount in situ hybridizations for isl1 (A–F) and pax6b (G–J) mRNA in control (A, C, E, G, I) and in isl1 −/− embryos (B, D, F, H, J) at 24 hpf (A, B, G, H), 48 hpf (C, D) and 76 hpf (E, F, I, J). Note that the pattern and intensity of isl1 signals in the pancreatic mesenchyme (black arrow head) and in the endocrine islet (white arrow head) is indistinguishable in control and mutant embryos. Embryos are shown from ventral with anterior to the left. (K–P) Whole mount in situ hybridizations for arx . (K, N) Dorsal view of control embryos at 24 hpf (K) and 48 hpf (N). Insets mark the position of the pancreatic area shown in higher magnification panels (L M, O, P). Images show a ventral view of control (L, O) and isl1 −/− mutant (M, P) embryos. (Q) qPCR analysis for hormone and transcription factor encoding mRNA of isolated pancreata from 96 hpf old embryos. Shown are relative expression levels in pancreata of mutant as compared to control embryos, normalized to the relative expression levels of ins obtained for 96 hpf whole embryonic mRNA.
    Figure Legend Snippet: Unchanged expression of early endocrine markers in isl1 mutants. Whole mount in situ hybridizations for isl1 (A–F) and pax6b (G–J) mRNA in control (A, C, E, G, I) and in isl1 −/− embryos (B, D, F, H, J) at 24 hpf (A, B, G, H), 48 hpf (C, D) and 76 hpf (E, F, I, J). Note that the pattern and intensity of isl1 signals in the pancreatic mesenchyme (black arrow head) and in the endocrine islet (white arrow head) is indistinguishable in control and mutant embryos. Embryos are shown from ventral with anterior to the left. (K–P) Whole mount in situ hybridizations for arx . (K, N) Dorsal view of control embryos at 24 hpf (K) and 48 hpf (N). Insets mark the position of the pancreatic area shown in higher magnification panels (L M, O, P). Images show a ventral view of control (L, O) and isl1 −/− mutant (M, P) embryos. (Q) qPCR analysis for hormone and transcription factor encoding mRNA of isolated pancreata from 96 hpf old embryos. Shown are relative expression levels in pancreata of mutant as compared to control embryos, normalized to the relative expression levels of ins obtained for 96 hpf whole embryonic mRNA.

    Techniques Used: Expressing, In Situ, Mutagenesis, Real-time Polymerase Chain Reaction, Isolation

    25) Product Images from "Antiviral signalling in human IPSC-derived neurons recapitulates neurodevelopmental disorder phenotypes"

    Article Title: Antiviral signalling in human IPSC-derived neurons recapitulates neurodevelopmental disorder phenotypes

    Journal: bioRxiv

    doi: 10.1101/789321

    PML bodies are persistently increased following treatment of NPCs with IFNγ, regulate transcription of MHCI genes and are spatially associated with HLA-B transcription. (A) Confocal images of PML nuclear bodies in untreated (D18 U) and IFNγ-treated (D18 T) NPCs. (B) Quantification of PML bodies per nucleus in D18 NPCs. D18 U: n = 354 cells; D18 T: n = 286 cells; 3 control cell lines. Two-tailed Mann-Whitney test. (C) Confocal images of nuclear PML bodies in untreated (D30 UU), pre-treated (D30 TU), acutely treated (D30 UT), and double-treated (D30 TT) D30 neurons. (D) Quantification of nuclear PML bodies in D30 neurons. D30 UU: n = 236 cells; D30 TU: n = 264 cells; D30 UT: n = 231 cells; D30 TT: n = 307 cells; 3 control cell lines. (E) Schematic representation of IFNγ and As 2 O 3 treatment conditions. Kruskal-Wallis test with Dunn’s multiple comparison test. (F and G) Quantification of PML bodies in D18 and D30 ‘UNTR’, ‘IFNγ’, ‘As’ and ‘As + IFNγ’ conditions. D18 UNTR: n = 57 cells; D18 IFNγ: n = 65 cells; D18 As: n = 64 cells; D18 IFNγ + As: n = 54 cells. D30 UNTR: n = 104 cells; D30 IFNγ: n = 129 cells; D30 As: n =119 cells; D30 As + IFNγ: n = 105 cells; 3 control cell lines. Kruskal-Wallis test with Dunn’s multiple comparison test. (H) qPCR analysis of HLA-B, HLA-C and B2M relative expression in D18 ‘UNTR’, ‘IFNγ’, ‘As’ and ‘As + IFNγ’ conditions. n = 4 biological replicates from 3 control cell lines. One-way ANOVA with Tukey’s multiple comparison test. (I) Confocal images of HLA-B RNAScope™ F.I.S.H. and PML immunocytochemistry in D18 ‘UNTR’, ‘IFNγ’, ‘As’ and ‘As + IFNγ’ conditions. (J and K) Quantification of HLA-B RNAScope™ integrated density and spots per nucleus in D18 ‘UNTR’, ‘IFNγ’, ‘As’ and ‘As + IFNγ’ conditions. D18 UNTR: n = 57 cells; D18 IFNγ: n = 65 cells; D18 As: n = 64 cells; D18 IFNγ + As: n = 54 cells. Kruskal-Wallis test with Dunn’s multiple comparison test. (L and M) Correlation analysis of HLA-B RNAScope and PML integrated density and spots per nucleus in D18 IFNγ-treated cells. n = 65 cells from 3 control cell lines. Linear regression analysis. (N) Confocal image of HLA-B RNAScope™ F.I.S.H. and PML immunocytochemistry in D30 IFNγ treatment condition. Arrow shows colocalisation between PML and HLA-B pre-mRNA. (O and P) Quantification of PML spots per micron 2 in whole nuclei, HLA-B RNAScope spots and HLA-B RNAScope spot perimeters in D18 NPCs and D30 neurons, IFNγ treatment condition. D18 IFNγ: n = 67 cells; D30 IFNγ: n = 58 cells; 3 control cell lines. Kruskal-Wallis test with Dunn’s multiple comparison test. (Q and R) DiAna distance analysis of centre-centre distances from HLA-B RNAScope spots to PML spots in real images and following random shuffle of PML and RNAScope spots, using nuclei as a bounding box. n = 405 measurements; 3 control cell lines. Wilcoxon matched-pairs test. Results are presented as means +/- SEM. * P
    Figure Legend Snippet: PML bodies are persistently increased following treatment of NPCs with IFNγ, regulate transcription of MHCI genes and are spatially associated with HLA-B transcription. (A) Confocal images of PML nuclear bodies in untreated (D18 U) and IFNγ-treated (D18 T) NPCs. (B) Quantification of PML bodies per nucleus in D18 NPCs. D18 U: n = 354 cells; D18 T: n = 286 cells; 3 control cell lines. Two-tailed Mann-Whitney test. (C) Confocal images of nuclear PML bodies in untreated (D30 UU), pre-treated (D30 TU), acutely treated (D30 UT), and double-treated (D30 TT) D30 neurons. (D) Quantification of nuclear PML bodies in D30 neurons. D30 UU: n = 236 cells; D30 TU: n = 264 cells; D30 UT: n = 231 cells; D30 TT: n = 307 cells; 3 control cell lines. (E) Schematic representation of IFNγ and As 2 O 3 treatment conditions. Kruskal-Wallis test with Dunn’s multiple comparison test. (F and G) Quantification of PML bodies in D18 and D30 ‘UNTR’, ‘IFNγ’, ‘As’ and ‘As + IFNγ’ conditions. D18 UNTR: n = 57 cells; D18 IFNγ: n = 65 cells; D18 As: n = 64 cells; D18 IFNγ + As: n = 54 cells. D30 UNTR: n = 104 cells; D30 IFNγ: n = 129 cells; D30 As: n =119 cells; D30 As + IFNγ: n = 105 cells; 3 control cell lines. Kruskal-Wallis test with Dunn’s multiple comparison test. (H) qPCR analysis of HLA-B, HLA-C and B2M relative expression in D18 ‘UNTR’, ‘IFNγ’, ‘As’ and ‘As + IFNγ’ conditions. n = 4 biological replicates from 3 control cell lines. One-way ANOVA with Tukey’s multiple comparison test. (I) Confocal images of HLA-B RNAScope™ F.I.S.H. and PML immunocytochemistry in D18 ‘UNTR’, ‘IFNγ’, ‘As’ and ‘As + IFNγ’ conditions. (J and K) Quantification of HLA-B RNAScope™ integrated density and spots per nucleus in D18 ‘UNTR’, ‘IFNγ’, ‘As’ and ‘As + IFNγ’ conditions. D18 UNTR: n = 57 cells; D18 IFNγ: n = 65 cells; D18 As: n = 64 cells; D18 IFNγ + As: n = 54 cells. Kruskal-Wallis test with Dunn’s multiple comparison test. (L and M) Correlation analysis of HLA-B RNAScope and PML integrated density and spots per nucleus in D18 IFNγ-treated cells. n = 65 cells from 3 control cell lines. Linear regression analysis. (N) Confocal image of HLA-B RNAScope™ F.I.S.H. and PML immunocytochemistry in D30 IFNγ treatment condition. Arrow shows colocalisation between PML and HLA-B pre-mRNA. (O and P) Quantification of PML spots per micron 2 in whole nuclei, HLA-B RNAScope spots and HLA-B RNAScope spot perimeters in D18 NPCs and D30 neurons, IFNγ treatment condition. D18 IFNγ: n = 67 cells; D30 IFNγ: n = 58 cells; 3 control cell lines. Kruskal-Wallis test with Dunn’s multiple comparison test. (Q and R) DiAna distance analysis of centre-centre distances from HLA-B RNAScope spots to PML spots in real images and following random shuffle of PML and RNAScope spots, using nuclei as a bounding box. n = 405 measurements; 3 control cell lines. Wilcoxon matched-pairs test. Results are presented as means +/- SEM. * P

    Techniques Used: Two Tailed Test, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Expressing, Immunocytochemistry

    26) Product Images from "Cyclin A triggers Mitosis either via Greatwall or Cyclin B"

    Article Title: Cyclin A triggers Mitosis either via Greatwall or Cyclin B

    Journal: bioRxiv

    doi: 10.1101/501684

    Cell proliferation in the absence of Cyclin A2, and B-type Cyclins. (A) Cell cycle analysis 24 hours after DIA treatment. Cells were analysed by EdU labelling, PI staining and FACS analysis. The histograms show the PI intensities while the dot plots show EdU incorporation (Y-axis) vs PI intensity (X-axis). Quantifications of these representative data involving three independent experiments are shown in Figure 1C . (B) Cell proliferation of A2 dd , CycB1 dd and B1 dd /B2 ko following mock or DIA treatment. 1000 cells were plated in 6 well plates and incubated for 10 days before methanol fixation and Crytsal Violet staining. (C) qPCR analysis of Cyclin B3 mRNA levels, following 72 hours depletion in B1 dd /B2 ko cells. For quantificatioon we used primers to amplify two control mRNAs, TATA binding protein, and Actin. The plot shows the levels of CycB3 siRNA depleted mRNA relative to Ctr siRNA transfected cells. (D) Mitotic index measurements of B1 dd /B2 ko cells with the indicated treatemnts. Cells were transfected with siRNA for 72 hours blocked for 24 hours with Thymidine fixed 12 hours after release from Thymidine. Protame and Apcin were added for the final two hours before fixation. Mitotic cells were scored based DAPI staining and on condensed chromosome formation, The bar-plots show mean values of three biological releats (n=100 per repast and sample), Error bars indicate standard deviation.
    Figure Legend Snippet: Cell proliferation in the absence of Cyclin A2, and B-type Cyclins. (A) Cell cycle analysis 24 hours after DIA treatment. Cells were analysed by EdU labelling, PI staining and FACS analysis. The histograms show the PI intensities while the dot plots show EdU incorporation (Y-axis) vs PI intensity (X-axis). Quantifications of these representative data involving three independent experiments are shown in Figure 1C . (B) Cell proliferation of A2 dd , CycB1 dd and B1 dd /B2 ko following mock or DIA treatment. 1000 cells were plated in 6 well plates and incubated for 10 days before methanol fixation and Crytsal Violet staining. (C) qPCR analysis of Cyclin B3 mRNA levels, following 72 hours depletion in B1 dd /B2 ko cells. For quantificatioon we used primers to amplify two control mRNAs, TATA binding protein, and Actin. The plot shows the levels of CycB3 siRNA depleted mRNA relative to Ctr siRNA transfected cells. (D) Mitotic index measurements of B1 dd /B2 ko cells with the indicated treatemnts. Cells were transfected with siRNA for 72 hours blocked for 24 hours with Thymidine fixed 12 hours after release from Thymidine. Protame and Apcin were added for the final two hours before fixation. Mitotic cells were scored based DAPI staining and on condensed chromosome formation, The bar-plots show mean values of three biological releats (n=100 per repast and sample), Error bars indicate standard deviation.

    Techniques Used: Cell Cycle Assay, Staining, FACS, Incubation, Real-time Polymerase Chain Reaction, Binding Assay, Transfection, Standard Deviation

    27) Product Images from "Globin mRNA reduction for whole-blood transcriptome sequencing"

    Article Title: Globin mRNA reduction for whole-blood transcriptome sequencing

    Journal: Scientific Reports

    doi: 10.1038/srep31584

    The effective range and limitations. ( a ) qPCR cycle threshold values are presented to indicate the number of required PCR cycles if less than 100 ng of whole-blood RNA is GL treated. ( b ) GL fold change reduction effect was measured by qPCR for three whole-blood RNA inputs: 1, 50 and 100 ng. ( c ) Scatter plot to measure the GL specificity based on an ERCC 92 mRNA spike-in mix over five different GL oligonucleotides compared with a GL-negative control. ( d ) The importance of the GL T n tail for masking. Different GL oligonucleotides with variable lengths of T n tails were analyzed, and the reduction effect of globin α was compared with the GL-negative experiment using qPCR.
    Figure Legend Snippet: The effective range and limitations. ( a ) qPCR cycle threshold values are presented to indicate the number of required PCR cycles if less than 100 ng of whole-blood RNA is GL treated. ( b ) GL fold change reduction effect was measured by qPCR for three whole-blood RNA inputs: 1, 50 and 100 ng. ( c ) Scatter plot to measure the GL specificity based on an ERCC 92 mRNA spike-in mix over five different GL oligonucleotides compared with a GL-negative control. ( d ) The importance of the GL T n tail for masking. Different GL oligonucleotides with variable lengths of T n tails were analyzed, and the reduction effect of globin α was compared with the GL-negative experiment using qPCR.

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

    The nature and reduction effect of GlobinLock. ( a ) Schematic globin mRNA with possible masking oligonucleotides and anchored oligo-T primer. All oligo sequences and modifications are shown in Supplementary Table 1 . ( b ) Five GL conditions and GL-negative controls were compared to measure globin α reduction fold change by qPCR. Template dilutions (10×) were used in this relative qPCR design and therefore the reduction effect up to ten is measured accurately according to existing dilution factor but fold change values above ten are out of the reported quantification range. ( c ) 3′-DNA long GL concentration effect on human globin α and β as measured by qPCR.
    Figure Legend Snippet: The nature and reduction effect of GlobinLock. ( a ) Schematic globin mRNA with possible masking oligonucleotides and anchored oligo-T primer. All oligo sequences and modifications are shown in Supplementary Table 1 . ( b ) Five GL conditions and GL-negative controls were compared to measure globin α reduction fold change by qPCR. Template dilutions (10×) were used in this relative qPCR design and therefore the reduction effect up to ten is measured accurately according to existing dilution factor but fold change values above ten are out of the reported quantification range. ( c ) 3′-DNA long GL concentration effect on human globin α and β as measured by qPCR.

    Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay

    28) Product Images from "Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B"

    Article Title: Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B

    Journal: The EMBO Journal

    doi: 10.15252/embj.2020104419

    Cell proliferation in the absence of cyclin A2 and B‐type cyclins Cyclin B2 knock‐out and induced degradation by immunoblotting. Indicated cell lines were analysed 24 h after mock or Dox/IAA/Asv (DIA) treatment using the indicated antibodies to confirm homozygous gene tagging and efficiency of protein degradation. Cell cycle analysis 24 h after DIA treatment. Cells were analysed by EdU labelling, PI staining and FACS analysis. The histograms show the PI intensities while the dot plots show EdU incorporation ( y ‐axis) vs PI intensity ( x ‐axis). Gating of cell cycle phases is indicated by colour (G1—black, S phase—green, G2/M phase—orange) Cell cycle phase frequencies quantified from flow cytometry data (shown in (B)) in indicated cell lines 24 h after DIA treatment, ( n = 3 experiments, s.d. indicated by error bars). Cell proliferation of A2 dd , CycB1 dd and B1 dd /B2 ko following mock or DIA treatment. One thousand cells were plated in each well (diameter 3.5 cm) and incubated for 10 days before methanol fixation and Crystal Violet staining. Kinetics of mitotic entry as measured by time‐lapse microscopy in A2 dd and B1 dd /B2 ko cells following mock or 4‐h DIA treatment of asynchronous cells. The cells were imaged for 16 h with 5‐min intervals using widefield DIC; mitotic entry was manually scored by detecting cell rounding. Curves display the cumulative mitotic index (data from three repeats, n > 500 cells per condition, s.d. indicated by shaded area). qPCR analysis of cyclin B3 mRNA levels, following 72‐h depletion in B1 dd /B2 ko cells. For quantification, we used primers to amplify two control mRNAs, TATA‐binding protein and actin. The plot shows the levels of CycB3 siRNA‐depleted mRNA relative to Ctr siRNA‐transfected cells. (Bars indicate the mean of three independent experiments; error bars indicate the standard deviation between these three repeats.) Mitotic index measurements of B1 dd /B2 ko cells with the indicated treatments. Cells were transfected with siRNA for 72 h; after 36 h, they were blocked for 24 h with Thymidine and fixed 12 h after release from Thymidine. ProTAME and Apcin were added for the final 2 h before fixation. Mitotic cells were scored based on DAPI staining and on condensed chromosome formation. The bar plots show mean values of three biological repeats ( n = 100 per repeat and sample, error bars indicate standard deviation, and P ‐values were calculated using an independent two‐sample t‐test). Cyclin A2 siRNA depletion in MCF7 and MCF10A cells. The cells were transfected with Ctr or cyclin A2 siRNA for indicated length of time and probed for cyclin A2 levels by immunoblotting. Cyclin A2 siRNA depletion causes endoreplication. Following 72 h of siRNA transfection, MCF7 and MCF10A cells were analysed by PI staining and FACS. The histograms show the changes in DNA content (PI Int.) towards > 4N following cyclin A2 depletion. Cyclin A2 siRNA and degron depletion in RPE‐1 cells. RPE‐1 OsTIR1 and RPE‐1 A2 dd cells were subjected to 72 h of cyclin A2 siRNA depletion and/or of DIA treatment as indicated and probed for cyclin A2 levels by immunoblotting. The longer exposure (L.E.) reveals incomplete depletion of cyclin 2 by siRNA. Cyclin A degron depletion causes accumulation of cells in G2 phase. Following 72 h of siRNA transfection or DIA treatment, RPE‐1 OsTIR1 and A2 dd cells were analysed by PI staining and FACS. The histograms show the changes in DNA content (PI Int.) towards > 4N following cyclin siRNA A2 depletion in RPE‐1 OsTIR1 cells, while DIA treatment in A2 dd cells does cause an increase in the 4N but not > 4N peak.
    Figure Legend Snippet: Cell proliferation in the absence of cyclin A2 and B‐type cyclins Cyclin B2 knock‐out and induced degradation by immunoblotting. Indicated cell lines were analysed 24 h after mock or Dox/IAA/Asv (DIA) treatment using the indicated antibodies to confirm homozygous gene tagging and efficiency of protein degradation. Cell cycle analysis 24 h after DIA treatment. Cells were analysed by EdU labelling, PI staining and FACS analysis. The histograms show the PI intensities while the dot plots show EdU incorporation ( y ‐axis) vs PI intensity ( x ‐axis). Gating of cell cycle phases is indicated by colour (G1—black, S phase—green, G2/M phase—orange) Cell cycle phase frequencies quantified from flow cytometry data (shown in (B)) in indicated cell lines 24 h after DIA treatment, ( n = 3 experiments, s.d. indicated by error bars). Cell proliferation of A2 dd , CycB1 dd and B1 dd /B2 ko following mock or DIA treatment. One thousand cells were plated in each well (diameter 3.5 cm) and incubated for 10 days before methanol fixation and Crystal Violet staining. Kinetics of mitotic entry as measured by time‐lapse microscopy in A2 dd and B1 dd /B2 ko cells following mock or 4‐h DIA treatment of asynchronous cells. The cells were imaged for 16 h with 5‐min intervals using widefield DIC; mitotic entry was manually scored by detecting cell rounding. Curves display the cumulative mitotic index (data from three repeats, n > 500 cells per condition, s.d. indicated by shaded area). qPCR analysis of cyclin B3 mRNA levels, following 72‐h depletion in B1 dd /B2 ko cells. For quantification, we used primers to amplify two control mRNAs, TATA‐binding protein and actin. The plot shows the levels of CycB3 siRNA‐depleted mRNA relative to Ctr siRNA‐transfected cells. (Bars indicate the mean of three independent experiments; error bars indicate the standard deviation between these three repeats.) Mitotic index measurements of B1 dd /B2 ko cells with the indicated treatments. Cells were transfected with siRNA for 72 h; after 36 h, they were blocked for 24 h with Thymidine and fixed 12 h after release from Thymidine. ProTAME and Apcin were added for the final 2 h before fixation. Mitotic cells were scored based on DAPI staining and on condensed chromosome formation. The bar plots show mean values of three biological repeats ( n = 100 per repeat and sample, error bars indicate standard deviation, and P ‐values were calculated using an independent two‐sample t‐test). Cyclin A2 siRNA depletion in MCF7 and MCF10A cells. The cells were transfected with Ctr or cyclin A2 siRNA for indicated length of time and probed for cyclin A2 levels by immunoblotting. Cyclin A2 siRNA depletion causes endoreplication. Following 72 h of siRNA transfection, MCF7 and MCF10A cells were analysed by PI staining and FACS. The histograms show the changes in DNA content (PI Int.) towards > 4N following cyclin A2 depletion. Cyclin A2 siRNA and degron depletion in RPE‐1 cells. RPE‐1 OsTIR1 and RPE‐1 A2 dd cells were subjected to 72 h of cyclin A2 siRNA depletion and/or of DIA treatment as indicated and probed for cyclin A2 levels by immunoblotting. The longer exposure (L.E.) reveals incomplete depletion of cyclin 2 by siRNA. Cyclin A degron depletion causes accumulation of cells in G2 phase. Following 72 h of siRNA transfection or DIA treatment, RPE‐1 OsTIR1 and A2 dd cells were analysed by PI staining and FACS. The histograms show the changes in DNA content (PI Int.) towards > 4N following cyclin siRNA A2 depletion in RPE‐1 OsTIR1 cells, while DIA treatment in A2 dd cells does cause an increase in the 4N but not > 4N peak.

    Techniques Used: Knock-Out, Cell Cycle Assay, Staining, FACS, Flow Cytometry, Incubation, Time-lapse Microscopy, Real-time Polymerase Chain Reaction, Binding Assay, Transfection, Standard Deviation

    29) Product Images from "Efficacy of IFN-?1 to Protect Human Airway Epithelial Cells against Human Rhinovirus 1B Infection"

    Article Title: Efficacy of IFN-?1 to Protect Human Airway Epithelial Cells against Human Rhinovirus 1B Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095134

    Priming effect of IFNs on HRV1B-induced interferon response. A549 were first treated with IFNs for 18-treatment approaches. After that cells were infected with HRV1B for 4 h in the same medium. After the infection period, either same medium was maintained on the cells (continuous) or replaced by fresh medium (pre-treated). Thereafter, cells were incubated for another 24 h and then collected for further analyses. mRNA levels of IFN-β (A) and IFN-λ1 (B) were determined by qPCR and fold changes were calculated with the 2 −ΔΔCt method. HRV1B induced average IFN-β expression was 1.8 (continuous) and 0.99 (pre-treated) folds and average IFN-λ1 expression was 1.92 (continuous) and 0.90 folds (pre-treated). *, p
    Figure Legend Snippet: Priming effect of IFNs on HRV1B-induced interferon response. A549 were first treated with IFNs for 18-treatment approaches. After that cells were infected with HRV1B for 4 h in the same medium. After the infection period, either same medium was maintained on the cells (continuous) or replaced by fresh medium (pre-treated). Thereafter, cells were incubated for another 24 h and then collected for further analyses. mRNA levels of IFN-β (A) and IFN-λ1 (B) were determined by qPCR and fold changes were calculated with the 2 −ΔΔCt method. HRV1B induced average IFN-β expression was 1.8 (continuous) and 0.99 (pre-treated) folds and average IFN-λ1 expression was 1.92 (continuous) and 0.90 folds (pre-treated). *, p

    Techniques Used: Infection, Incubation, Real-time Polymerase Chain Reaction, Expressing

    IFN-induced antiviral state protects against HRV1B infection in A549. A549 were treated with IFNs for 18(n = 6). Fold-changes were calculated with the 2 −ΔΔCt method (A). A549 were first treated with IFNs for 18 h. Next, according to the continuous and pre-treatment approaches, cells were infected with HRV1B and incubated for another 24 h (n = 6). After that, cells were collected for further analyses. Viral copies were determined by qPCR and fold-changes were calculated with the 2 −ΔΔCt method (B). *, p
    Figure Legend Snippet: IFN-induced antiviral state protects against HRV1B infection in A549. A549 were treated with IFNs for 18(n = 6). Fold-changes were calculated with the 2 −ΔΔCt method (A). A549 were first treated with IFNs for 18 h. Next, according to the continuous and pre-treatment approaches, cells were infected with HRV1B and incubated for another 24 h (n = 6). After that, cells were collected for further analyses. Viral copies were determined by qPCR and fold-changes were calculated with the 2 −ΔΔCt method (B). *, p

    Techniques Used: Infection, Incubation, Real-time Polymerase Chain Reaction

    IFNs-induced antiviral state protects against HRV1B infection in PBECs. PBECs were infected with HRV1B for 4(continuous) or replaced with fresh medium (pre-treated). After that cells were incubated for another 24 h. Then the supernatant was collected to determine TCID50 (C) while cells were collected to determine viral copies (A). PBECs were treated with IFNs for 18 h and then mRNA expression of different ISGs was determined by qPCR. Fold changes were calculated with the 2 −ΔΔCt method (B). *, p
    Figure Legend Snippet: IFNs-induced antiviral state protects against HRV1B infection in PBECs. PBECs were infected with HRV1B for 4(continuous) or replaced with fresh medium (pre-treated). After that cells were incubated for another 24 h. Then the supernatant was collected to determine TCID50 (C) while cells were collected to determine viral copies (A). PBECs were treated with IFNs for 18 h and then mRNA expression of different ISGs was determined by qPCR. Fold changes were calculated with the 2 −ΔΔCt method (B). *, p

    Techniques Used: Infection, Incubation, Expressing, Real-time Polymerase Chain Reaction

    Long-lasting antiviral state induced by IFNs. A549 were treated with IFNs for 18-treatment approaches, cells cultured for another 72 h and collected afterwards for further analyses (n = 4). mRNA expression of different genes was determined by qPCR and fold changes were calculated with the 2 −ΔΔCt method (A B). Moreover, to evaluate the antiviral status of the cells after 72 h of culturing, cells were infected with HRV1B and incubated for another 24 h (n = 5). After that, viral copies were determined by qPCR and fold changes were calculated with the 2 −ΔΔCt method (C). *, p
    Figure Legend Snippet: Long-lasting antiviral state induced by IFNs. A549 were treated with IFNs for 18-treatment approaches, cells cultured for another 72 h and collected afterwards for further analyses (n = 4). mRNA expression of different genes was determined by qPCR and fold changes were calculated with the 2 −ΔΔCt method (A B). Moreover, to evaluate the antiviral status of the cells after 72 h of culturing, cells were infected with HRV1B and incubated for another 24 h (n = 5). After that, viral copies were determined by qPCR and fold changes were calculated with the 2 −ΔΔCt method (C). *, p

    Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Infection, Incubation

    HRV1B induced interferon response in PBECs and its comparison with other stimuli. PBECs were infected with HRV1B for 4(n = 4). The mRNA expression of ISGs was determined with qPCR and fold changes were calculated with the 2 −ΔΔCt method (A). Cells were infected with RSV (MOI-1, 1 h) or HRV1B (4 h). After infection, virus-containing medium was removed and replaced with fresh medium. Additionally, cells were stimulated with poly(I:C)/LyoVec (500 ng/ml) for 24 h (n = 3). Then cells were collected and IFNβ/λ1 mRNA expression was determined by qPCR and relative amount of mRNA was calculated with the 2 −ΔCt method (B). *, p
    Figure Legend Snippet: HRV1B induced interferon response in PBECs and its comparison with other stimuli. PBECs were infected with HRV1B for 4(n = 4). The mRNA expression of ISGs was determined with qPCR and fold changes were calculated with the 2 −ΔΔCt method (A). Cells were infected with RSV (MOI-1, 1 h) or HRV1B (4 h). After infection, virus-containing medium was removed and replaced with fresh medium. Additionally, cells were stimulated with poly(I:C)/LyoVec (500 ng/ml) for 24 h (n = 3). Then cells were collected and IFNβ/λ1 mRNA expression was determined by qPCR and relative amount of mRNA was calculated with the 2 −ΔCt method (B). *, p

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

    HRV1B induced interferon response in A549. A549 were infected with HRV1B for 4(n = 6). The mRNA expression of ISGs was determined with qPCR and fold changes were calculated with the 2 −ΔΔCt method (A). Cells were infected with RSV (MOI-1, 1 h) or HRV1B (4 h). After infection, virus containing medium was removed and replaced with fresh medium. Then cells were collected after 24 h (n = 5). While with poly(I:C)/LyoVec (500 ng/ml), cells were collected after 18 h of stimulation (n = 3). IFNβ/λ1 mRNA expression was determined by qPCR and fold changes were calculated with the 2 −ΔΔCt method (B). *, p
    Figure Legend Snippet: HRV1B induced interferon response in A549. A549 were infected with HRV1B for 4(n = 6). The mRNA expression of ISGs was determined with qPCR and fold changes were calculated with the 2 −ΔΔCt method (A). Cells were infected with RSV (MOI-1, 1 h) or HRV1B (4 h). After infection, virus containing medium was removed and replaced with fresh medium. Then cells were collected after 24 h (n = 5). While with poly(I:C)/LyoVec (500 ng/ml), cells were collected after 18 h of stimulation (n = 3). IFNβ/λ1 mRNA expression was determined by qPCR and fold changes were calculated with the 2 −ΔΔCt method (B). *, p

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

    30) Product Images from "Cell surface receptor kinase FERONIA linked to nutrient sensor TORC1 signaling controls root hair growth at low temperature in Arabidopsis thaliana"

    Article Title: Cell surface receptor kinase FERONIA linked to nutrient sensor TORC1 signaling controls root hair growth at low temperature in Arabidopsis thaliana

    Journal: bioRxiv

    doi: 10.1101/2022.01.10.475584

    Quantitative PCR of TOR expression levels in Col-0 roots grown at 22°C and 10°C. ACT2 expression was used for normalization of gene expression. Three biological replicates and three technical replicates per experiment were performed.
    Figure Legend Snippet: Quantitative PCR of TOR expression levels in Col-0 roots grown at 22°C and 10°C. ACT2 expression was used for normalization of gene expression. Three biological replicates and three technical replicates per experiment were performed.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    31) Product Images from "Apolipoprotein E expression pattern in human induced pluripotent stem cells duringin vitro neural induction"

    Article Title: Apolipoprotein E expression pattern in human induced pluripotent stem cells duringin vitro neural induction

    Journal: F1000Research

    doi: 10.12688/f1000research.23580.1

    APOE gene expression changes according to the differentiation state of cells during in vitro directed differentiation. A ) Schematic diagram of directed differentiation. CTR_M3_36S iPSCs were maintained in stem cell maintenance medium after replating (D-2/-1). On D0 neural induction began by changing the stem cell maintenance medium to neural induction medium. N2:B27 was used from D8 onwards. Medium was changed every 24 hrs throughout the entire differentiation period. Neural passaging 1, 2, and 3 were carried out on D7, D12, and D15/16, respectively. Total RNA extraction was made on cells that were not used for neural passaging on D7, D12, D15/16, and D18/19. Neural induction medium composition for each differentiation lineage and N2:B27 medium composition are also shown. B ) APOE gene expression is reduced along neural induction regardless of lineage. Real-time qPCR was performed on CTR_M3_36S iPSCs undergoing directed differentiation at D7, D12, D15/16, and D18/19. APOE expression was normalised to that of GAPDH . D7 samples were used as reference samples for each lineage. One-way ANOVA with Bonferroni correction. n = 3. Mean (bars) with S.E.M. (error bars) shown. **** ANOVA p-value
    Figure Legend Snippet: APOE gene expression changes according to the differentiation state of cells during in vitro directed differentiation. A ) Schematic diagram of directed differentiation. CTR_M3_36S iPSCs were maintained in stem cell maintenance medium after replating (D-2/-1). On D0 neural induction began by changing the stem cell maintenance medium to neural induction medium. N2:B27 was used from D8 onwards. Medium was changed every 24 hrs throughout the entire differentiation period. Neural passaging 1, 2, and 3 were carried out on D7, D12, and D15/16, respectively. Total RNA extraction was made on cells that were not used for neural passaging on D7, D12, D15/16, and D18/19. Neural induction medium composition for each differentiation lineage and N2:B27 medium composition are also shown. B ) APOE gene expression is reduced along neural induction regardless of lineage. Real-time qPCR was performed on CTR_M3_36S iPSCs undergoing directed differentiation at D7, D12, D15/16, and D18/19. APOE expression was normalised to that of GAPDH . D7 samples were used as reference samples for each lineage. One-way ANOVA with Bonferroni correction. n = 3. Mean (bars) with S.E.M. (error bars) shown. **** ANOVA p-value

    Techniques Used: Expressing, In Vitro, Passaging, RNA Extraction, Real-time Polymerase Chain Reaction

    32) Product Images from "Combined Field Inoculations of Pseudomonas Bacteria, Arbuscular Mycorrhizal Fungi, and Entomopathogenic Nematodes and their Effects on Wheat Performance"

    Article Title: Combined Field Inoculations of Pseudomonas Bacteria, Arbuscular Mycorrhizal Fungi, and Entomopathogenic Nematodes and their Effects on Wheat Performance

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2017.01809

    Abundance of Rhizoglomus irregulare in wheat roots in the COMBINATION (A,B) and PERFORMANCE-2 (C) field trials. (A) In the COMBINATION experiment, R. irregulare strain INOQ TOP was inoculated comparing high (F1) vs. low (F1*) dosages, with one of the treatments including the AMF strain SAF22 (F2). (B) In the same experiment, R. irregulare INOQ TOP (F1) was quantified in combination with bacteria, i.e., Pseudomonas protegens CHA0-Rif (B1), and nematodes, i.e., Heterorhabditis bacteriophora Andermatt (N2). (C) In the PERFORMANCE-2 experiment, R. irregulare INOQ TOP at the lower dosage (F1*) was used for the combination treatments with bacterial mixture (BM; i.e., P. protegens + Pseudomonas chlororaphis ) and nematode mixture (NM; i.e., Heterorhabditis megidis + H. bacteriophora + Steinernema feltiae ; for details see Figure S1 ). Control, non-inoculated control; AMF control, substrate control for AMF inoculation. R. irregulare was measured with quantitative PCR employing species-specific primers developed by Alkan et al. ( 2006 ) for INOQ TOP or their modified variants with enhanced specificity for SAF22 (Bender et al., unpublished). Bar graphs report mean normalized ( R. irregulare relative to plant DNA) abundance (± SEM; COMBINATION, n = 4; PERFORMANCE-2, n = 7–9). Statistical analyses were performed on log-transformed data; asterisks and different letters indicate statistical significance at P
    Figure Legend Snippet: Abundance of Rhizoglomus irregulare in wheat roots in the COMBINATION (A,B) and PERFORMANCE-2 (C) field trials. (A) In the COMBINATION experiment, R. irregulare strain INOQ TOP was inoculated comparing high (F1) vs. low (F1*) dosages, with one of the treatments including the AMF strain SAF22 (F2). (B) In the same experiment, R. irregulare INOQ TOP (F1) was quantified in combination with bacteria, i.e., Pseudomonas protegens CHA0-Rif (B1), and nematodes, i.e., Heterorhabditis bacteriophora Andermatt (N2). (C) In the PERFORMANCE-2 experiment, R. irregulare INOQ TOP at the lower dosage (F1*) was used for the combination treatments with bacterial mixture (BM; i.e., P. protegens + Pseudomonas chlororaphis ) and nematode mixture (NM; i.e., Heterorhabditis megidis + H. bacteriophora + Steinernema feltiae ; for details see Figure S1 ). Control, non-inoculated control; AMF control, substrate control for AMF inoculation. R. irregulare was measured with quantitative PCR employing species-specific primers developed by Alkan et al. ( 2006 ) for INOQ TOP or their modified variants with enhanced specificity for SAF22 (Bender et al., unpublished). Bar graphs report mean normalized ( R. irregulare relative to plant DNA) abundance (± SEM; COMBINATION, n = 4; PERFORMANCE-2, n = 7–9). Statistical analyses were performed on log-transformed data; asterisks and different letters indicate statistical significance at P

    Techniques Used: Real-time Polymerase Chain Reaction, Modification, Transformation Assay

    End of the season presence of inoculant and resident entomopathogenic nematodes in the COMBINATION (A) , PERFORMANCE-1 (B) , and PERFORMANCE-2 (C) field trials. Four different EPN species Heterorhabditis megidis (N1), Heterorhabditis bacteriphora (N2), Steinernema carpocapsae (N3), and Steinernema feltiae (N4) were inoculated individually or in combination with Pseudomonas protegens (B1), Pseudomonas chlororaphis (B2) and Rhizoglomus irregularis at two dosages (F1 and F1*). Mixtures of EPN (N1+N2+N4) or of the two bacteria (B1+B2) are indicated with NM and BM, respectively (for details see Figure S1 ). To determine the persistence of the EPN in soil of the different nematode inoculants as well as the impact of each treatment on the resident population of entompathogenic nematodes (EPN), a DNA extraction procedure followed by a qPCR approach was performed. Data are expressed as total EPN 100 g −1 of dry soil. Bar graphs report means (± SEM) and pie-charts show the proportion of native EPN vs. augmented EPN. Significant differences between treatments were calculated with one-way ANOVA (significance level P
    Figure Legend Snippet: End of the season presence of inoculant and resident entomopathogenic nematodes in the COMBINATION (A) , PERFORMANCE-1 (B) , and PERFORMANCE-2 (C) field trials. Four different EPN species Heterorhabditis megidis (N1), Heterorhabditis bacteriphora (N2), Steinernema carpocapsae (N3), and Steinernema feltiae (N4) were inoculated individually or in combination with Pseudomonas protegens (B1), Pseudomonas chlororaphis (B2) and Rhizoglomus irregularis at two dosages (F1 and F1*). Mixtures of EPN (N1+N2+N4) or of the two bacteria (B1+B2) are indicated with NM and BM, respectively (for details see Figure S1 ). To determine the persistence of the EPN in soil of the different nematode inoculants as well as the impact of each treatment on the resident population of entompathogenic nematodes (EPN), a DNA extraction procedure followed by a qPCR approach was performed. Data are expressed as total EPN 100 g −1 of dry soil. Bar graphs report means (± SEM) and pie-charts show the proportion of native EPN vs. augmented EPN. Significant differences between treatments were calculated with one-way ANOVA (significance level P

    Techniques Used: DNA Extraction, Real-time Polymerase Chain Reaction

    33) Product Images from "Intestinal microbiome is related to lifetime antibiotic use in Finnish pre-school children"

    Article Title: Intestinal microbiome is related to lifetime antibiotic use in Finnish pre-school children

    Journal: Nature Communications

    doi: 10.1038/ncomms10410

    Macrolide resistance and bile-salt hydrolase abundance in relation to time since the last macrolide course. The dashed lines show the model fit (linear or polynomial), R 2 indicates the variation explained by the model and the P values (estimated using linear models) are indicated. ( a ) Macrolide resistance potential inferred from metagenomic analysis, N =14. ( b ) Bile-salt hydrolase abundance in metagenomes, N =14. ( c ) Macrolide resistance measured as proportion of anaerobic c.f.u.'s growing with erythromycin compared with c.f.u. without erythromycin, N =80. ( d ) Combined relative abundance of three bile-salt hydrolase genes ( bsh ) based on qPCR as a function of time since last macrolide course, N =37.
    Figure Legend Snippet: Macrolide resistance and bile-salt hydrolase abundance in relation to time since the last macrolide course. The dashed lines show the model fit (linear or polynomial), R 2 indicates the variation explained by the model and the P values (estimated using linear models) are indicated. ( a ) Macrolide resistance potential inferred from metagenomic analysis, N =14. ( b ) Bile-salt hydrolase abundance in metagenomes, N =14. ( c ) Macrolide resistance measured as proportion of anaerobic c.f.u.'s growing with erythromycin compared with c.f.u. without erythromycin, N =80. ( d ) Combined relative abundance of three bile-salt hydrolase genes ( bsh ) based on qPCR as a function of time since last macrolide course, N =37.

    Techniques Used: Real-time Polymerase Chain Reaction

    34) Product Images from "Intronic enhancer region governs transcript-specific Bdnf expression in rodent neurons"

    Article Title: Intronic enhancer region governs transcript-specific Bdnf expression in rodent neurons

    Journal: eLife

    doi: 10.7554/eLife.65161

    Verification of the deletion of the +3 kb enhancer region in mouse embryonic stem cell (mESC) clones. ( A ) Genomic positions of the gRNA targeting sequences that were used for deleting the +3 kb enhancer region with CRISPR/Cas9, PCR amplicons used for agarose gel electrophoresis-based genotyping (results not shown), and qPCR amplicons used to determine copy numbers of the indicated regions. ( B ) Genotypes of the mESC clones used in this study determined by qPCR. The table shows copy numbers for the indicated regions. Only clones with intact 5' and 3' distal flanking regions were selected, and their genotype was assigned based on the copy number of the enhancer core region. Regions showing a deletion of one or two alleles are indicated with pink and red, respectively.
    Figure Legend Snippet: Verification of the deletion of the +3 kb enhancer region in mouse embryonic stem cell (mESC) clones. ( A ) Genomic positions of the gRNA targeting sequences that were used for deleting the +3 kb enhancer region with CRISPR/Cas9, PCR amplicons used for agarose gel electrophoresis-based genotyping (results not shown), and qPCR amplicons used to determine copy numbers of the indicated regions. ( B ) Genotypes of the mESC clones used in this study determined by qPCR. The table shows copy numbers for the indicated regions. Only clones with intact 5' and 3' distal flanking regions were selected, and their genotype was assigned based on the copy number of the enhancer core region. Regions showing a deletion of one or two alleles are indicated with pink and red, respectively.

    Techniques Used: CRISPR, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Genotyping Assay, Real-time Polymerase Chain Reaction, Clone Assay

    35) Product Images from "Transcriptional profiling of human macrophages during infection with Bordetella pertussis"

    Article Title: Transcriptional profiling of human macrophages during infection with Bordetella pertussis

    Journal: RNA Biology

    doi: 10.1080/15476286.2020.1727694

    Validation of the RNA-seq results with quantitative PCR. (A) RT-qPCR analysis was performed to assay the relative expression profiles of IL10, IL23, SOCS3, NFKB1, STAT4, LAMP3, ADORA2A and BIRC3 genes in infected THP-1 macrophages. Relative gene expression was compared between infected and uninfected macrophages harvested 2, 6 and 24 h post-infection (pi). (B) RT-qPCR analysis was performed to assay the relative expression profiles of vag8, prn, vrg6 and BP2871 genes in intracellular B. pertussis cells. Relative gene expression was compared between intracellular and unexposed bacteria 2, 6 and 24 h post-infection (pi). Fold change (FC) values are means (bars) ± standard deviations (error bars) from three biological replicate experiments. Values depicted between the bars denote the fold changes in expression of the specific gene between corresponding time points deduced from RNA-seq results determined 2, 6 and 24 h pi. ND, not determined in the corresponding analysis.
    Figure Legend Snippet: Validation of the RNA-seq results with quantitative PCR. (A) RT-qPCR analysis was performed to assay the relative expression profiles of IL10, IL23, SOCS3, NFKB1, STAT4, LAMP3, ADORA2A and BIRC3 genes in infected THP-1 macrophages. Relative gene expression was compared between infected and uninfected macrophages harvested 2, 6 and 24 h post-infection (pi). (B) RT-qPCR analysis was performed to assay the relative expression profiles of vag8, prn, vrg6 and BP2871 genes in intracellular B. pertussis cells. Relative gene expression was compared between intracellular and unexposed bacteria 2, 6 and 24 h post-infection (pi). Fold change (FC) values are means (bars) ± standard deviations (error bars) from three biological replicate experiments. Values depicted between the bars denote the fold changes in expression of the specific gene between corresponding time points deduced from RNA-seq results determined 2, 6 and 24 h pi. ND, not determined in the corresponding analysis.

    Techniques Used: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Infection

    36) Product Images from "In vivo distribution of U87MG cells injected into the lateral ventricle of rats with spinal cord injury"

    Article Title: In vivo distribution of U87MG cells injected into the lateral ventricle of rats with spinal cord injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202307

    Quantitative real-time PCR (qPCR) against human specific sequence. To detect U87MGs in the CNS of rats with SCI, each region of the CNS was analyzed by qPCR using human-specific primers. (A) Relative amounts of human Alu sequence were quantified and compared. Height = Average, Error bar = Standard deviation. *, P
    Figure Legend Snippet: Quantitative real-time PCR (qPCR) against human specific sequence. To detect U87MGs in the CNS of rats with SCI, each region of the CNS was analyzed by qPCR using human-specific primers. (A) Relative amounts of human Alu sequence were quantified and compared. Height = Average, Error bar = Standard deviation. *, P

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

    37) Product Images from "Zebularine induces enzymatic DNA–protein crosslinks in 45S rDNA heterochromatin of Arabidopsis nuclei"

    Article Title: Zebularine induces enzymatic DNA–protein crosslinks in 45S rDNA heterochromatin of Arabidopsis nuclei

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab1218

    Zebularine induces DPCs with MET1. N-ChIP followed by qPCR was used to quantify binding of MET1-RFP using anti-RFP antibody at 45S rDNA and 5S rDNA . Binding to a nontarget locus ( ACT2 ) was evaluated to analyze binding specificity. Arabidopsis seedlings were treated (T) as mock (CTRL) or with 40 μM zebularine (ZEB) for 24 h with 0, 48 and 144 h of recovery (R) at zebularine-free conditions. Significance was determined by comparing IgG and MET1-RFP signals within the same experimental point and also between control and zebularine-treated samples. *** P
    Figure Legend Snippet: Zebularine induces DPCs with MET1. N-ChIP followed by qPCR was used to quantify binding of MET1-RFP using anti-RFP antibody at 45S rDNA and 5S rDNA . Binding to a nontarget locus ( ACT2 ) was evaluated to analyze binding specificity. Arabidopsis seedlings were treated (T) as mock (CTRL) or with 40 μM zebularine (ZEB) for 24 h with 0, 48 and 144 h of recovery (R) at zebularine-free conditions. Significance was determined by comparing IgG and MET1-RFP signals within the same experimental point and also between control and zebularine-treated samples. *** P

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

    38) Product Images from "The Effects of Alpha-Linolenic Acid on the Secretory Activity of Astrocytes and β Amyloid-Associated Neurodegeneration in Differentiated SH-SY5Y Cells: Alpha-Linolenic Acid Protects the SH-SY5Y cells against β Amyloid Toxicity"

    Article Title: The Effects of Alpha-Linolenic Acid on the Secretory Activity of Astrocytes and β Amyloid-Associated Neurodegeneration in Differentiated SH-SY5Y Cells: Alpha-Linolenic Acid Protects the SH-SY5Y cells against β Amyloid Toxicity

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2020/8908901

    The CM and ALA-CM pretreatment modulates Amyloid β - (A β 1-42 -) induced effects on mitophagy and autophagy. On the day 6th, the SH-SY5Y cells (differentiated) were pretreated for 1 h with CM or ALA-CM before the addition of 5 μ M A β 1-42 for the next 24 h. The SH-SY5Y cells were also exposed to the cotreatment of CM and ALA-CM with Insulin Degrading Enzyme (IDE) to check whether insulin and IGF-I presence of CM and ALA-CM was responsible for the neuroprotective effect. Positive controls were the SH-SY5Y cells treated with insulin and carbonyl-cyano-m-chlorophenylhydrazone-CCCP (10 μ M). RT-qPCR results showed that A β 1-42 significantly increased mRNA levels of markers of mitophagy ( PINK-1 (a), PARKIN (b)), and autophagy ( ATG5 (c) and LC3β (d)). Whereas pretreatment with CM of the SH-SY5Y cells exposed to A β 1-42 significantly decreased expression of mitophagy and autophagy markers. IDE increased levels of mitophagy and autophagy markers. The immunocytofluorescence staining showed that the A β 1-42 treated cells had an increased PARKIN fluorescence intensity, a well-known marker of mitophagy (e, f). Whereas pretreatment with CM and ALA-CM of the SH-SY5Y cells exposed to A β 1-42 significantly decreased PARKIN fluorescence intensity. A similar effect was observed after the insulin treatment. IDE increased PARKIN fluorescence intensity. Bar graph showed the relative fluorescence intensity of PARKIN. Antibody against PARKIN was used to stain marker of mitophagy in differentiated SH-SY5Y cells (shown as green signals). Hoechst 33342 was used to stain nuclei (shown as blue signals). Scale bar is 20 μ m. The results showed that A β 1-42 exposure has a similar effect to CCCP suggesting that A β 1-42 induce mitophagy and autophagy. One-way ANOVA followed by Tukey's multiple comparisons test at the 0.05 level was used to determine differences between the treated cells and untreated control cells. Results are presented as means ± SEM ( n = 3 − 8). RT-qPCR fold increase and the fluorescence intensity were calculated according to the formula described in the Materials and Methods section. Statistical differences between the treated cells and untreated control cells are indicated by asterisks ( ∗ for P
    Figure Legend Snippet: The CM and ALA-CM pretreatment modulates Amyloid β - (A β 1-42 -) induced effects on mitophagy and autophagy. On the day 6th, the SH-SY5Y cells (differentiated) were pretreated for 1 h with CM or ALA-CM before the addition of 5 μ M A β 1-42 for the next 24 h. The SH-SY5Y cells were also exposed to the cotreatment of CM and ALA-CM with Insulin Degrading Enzyme (IDE) to check whether insulin and IGF-I presence of CM and ALA-CM was responsible for the neuroprotective effect. Positive controls were the SH-SY5Y cells treated with insulin and carbonyl-cyano-m-chlorophenylhydrazone-CCCP (10 μ M). RT-qPCR results showed that A β 1-42 significantly increased mRNA levels of markers of mitophagy ( PINK-1 (a), PARKIN (b)), and autophagy ( ATG5 (c) and LC3β (d)). Whereas pretreatment with CM of the SH-SY5Y cells exposed to A β 1-42 significantly decreased expression of mitophagy and autophagy markers. IDE increased levels of mitophagy and autophagy markers. The immunocytofluorescence staining showed that the A β 1-42 treated cells had an increased PARKIN fluorescence intensity, a well-known marker of mitophagy (e, f). Whereas pretreatment with CM and ALA-CM of the SH-SY5Y cells exposed to A β 1-42 significantly decreased PARKIN fluorescence intensity. A similar effect was observed after the insulin treatment. IDE increased PARKIN fluorescence intensity. Bar graph showed the relative fluorescence intensity of PARKIN. Antibody against PARKIN was used to stain marker of mitophagy in differentiated SH-SY5Y cells (shown as green signals). Hoechst 33342 was used to stain nuclei (shown as blue signals). Scale bar is 20 μ m. The results showed that A β 1-42 exposure has a similar effect to CCCP suggesting that A β 1-42 induce mitophagy and autophagy. One-way ANOVA followed by Tukey's multiple comparisons test at the 0.05 level was used to determine differences between the treated cells and untreated control cells. Results are presented as means ± SEM ( n = 3 − 8). RT-qPCR fold increase and the fluorescence intensity were calculated according to the formula described in the Materials and Methods section. Statistical differences between the treated cells and untreated control cells are indicated by asterisks ( ∗ for P

    Techniques Used: Quantitative RT-PCR, Expressing, Staining, Fluorescence, Marker

    The CM and ALA-CM pretreatment reversed Amyloid β - (A β 1-42 -) induced synaptic toxicity in differentiated SH-SY5Y cells. On the day 6th, the SH-SY5Y cells (differentiated) were pretreated for 1 h with CM or ALA-CM before the addition of 5 μ M A β 1-42 for the next 24 h. The SH-SY5Y cells were also exposed to the cotreatment of CM and ALA-CM with Insulin Degrading Enzyme (IDE) to check whether insulin and IGF-I presence in CM and ALA-CM was responsible for the neuroprotective effect. Positive controls were the SH-SY5Y cells treated with insulin and carbonyl-cyano-m-chlorophenylhydrazone-CCCP (10 μ M). RT-qPCR results indicated that A β 1-42 significantly decreased mRNA levels of Synaptophysin (a) and PSD95 (b), well-known synaptic markers. The CM and ALA-CM pretreatment reversed the effect of A β 1-42 when compared with DM + A β 1−42 group. The IDE treatment of CM and ALA-CM reduced this effect. The immunocytofluorescence staining showed that the A β 1-42 treated cells had a decreased Synaptophysin (c, e) and TUJ 1 ( β 3-Tubulin) (d, f) fluorescence intensity and increased neurites fragmentation. Moreover, results showed that the CM and ALA-CM pretreatment reversed the A β 1-42 –induced synaptic toxicity in differentiated SH-SY5Y cells. A similar effect of TUJ 1 fluorescence intensity was observed after the treatment of differentiated SH-SY5Y cells with insulin (d, f). The IDE treatment of CM and ALA-CM reduced this effect. The cells were subjected to immunocytofluorescence staining with antibodies against Synaptophysin and TUJ 1. TUJ 1 was used as a marker to stain differentiated SH-SY5Y cells (show as green signals). Synaptophysin was used to stain synaptic in differentiated SH-SY5Y cells (show as red signals). Hoechst 33342 was used to stain nuclei (show as blue signals) (see Materials and Methods section). Bar graphs (e, f) showed the relative fluorescence intensity of Synaptophysin and TUJ 1. Scale bar is 20 μ m. One-way ANOVA followed by Tukey's multiple comparisons test at the 0.05 level was used to determine differences between the treated cells and untreated control cells. Results are presented as means ± SEM ( n = 3 − 8). RT-qPCR fold increase and the fluorescence intensity were calculated according to the formula described in the Materials and Methods section. Statistical differences between the treated group and untreated control cells are indicated by asterisks ( ∗ for P
    Figure Legend Snippet: The CM and ALA-CM pretreatment reversed Amyloid β - (A β 1-42 -) induced synaptic toxicity in differentiated SH-SY5Y cells. On the day 6th, the SH-SY5Y cells (differentiated) were pretreated for 1 h with CM or ALA-CM before the addition of 5 μ M A β 1-42 for the next 24 h. The SH-SY5Y cells were also exposed to the cotreatment of CM and ALA-CM with Insulin Degrading Enzyme (IDE) to check whether insulin and IGF-I presence in CM and ALA-CM was responsible for the neuroprotective effect. Positive controls were the SH-SY5Y cells treated with insulin and carbonyl-cyano-m-chlorophenylhydrazone-CCCP (10 μ M). RT-qPCR results indicated that A β 1-42 significantly decreased mRNA levels of Synaptophysin (a) and PSD95 (b), well-known synaptic markers. The CM and ALA-CM pretreatment reversed the effect of A β 1-42 when compared with DM + A β 1−42 group. The IDE treatment of CM and ALA-CM reduced this effect. The immunocytofluorescence staining showed that the A β 1-42 treated cells had a decreased Synaptophysin (c, e) and TUJ 1 ( β 3-Tubulin) (d, f) fluorescence intensity and increased neurites fragmentation. Moreover, results showed that the CM and ALA-CM pretreatment reversed the A β 1-42 –induced synaptic toxicity in differentiated SH-SY5Y cells. A similar effect of TUJ 1 fluorescence intensity was observed after the treatment of differentiated SH-SY5Y cells with insulin (d, f). The IDE treatment of CM and ALA-CM reduced this effect. The cells were subjected to immunocytofluorescence staining with antibodies against Synaptophysin and TUJ 1. TUJ 1 was used as a marker to stain differentiated SH-SY5Y cells (show as green signals). Synaptophysin was used to stain synaptic in differentiated SH-SY5Y cells (show as red signals). Hoechst 33342 was used to stain nuclei (show as blue signals) (see Materials and Methods section). Bar graphs (e, f) showed the relative fluorescence intensity of Synaptophysin and TUJ 1. Scale bar is 20 μ m. One-way ANOVA followed by Tukey's multiple comparisons test at the 0.05 level was used to determine differences between the treated cells and untreated control cells. Results are presented as means ± SEM ( n = 3 − 8). RT-qPCR fold increase and the fluorescence intensity were calculated according to the formula described in the Materials and Methods section. Statistical differences between the treated group and untreated control cells are indicated by asterisks ( ∗ for P

    Techniques Used: Quantitative RT-PCR, Staining, Fluorescence, Marker

    39) Product Images from "Characterization of zebrafish (Danio rerio) muscle ankyrin repeat proteins reveals their conserved response to endurance exercise"

    Article Title: Characterization of zebrafish (Danio rerio) muscle ankyrin repeat proteins reveals their conserved response to endurance exercise

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0204312

    Expression of the zebrafish MARP genes in adult heart and skeletal muscle. Quantification of ankrd1a , ankrd1b and ankrd2 expression was done by qPCR, using mRNA isolated from the hearts of 8 fish, pooled in 4 groups and the skeletal muscles of 4 fish. Relative level of transcripts in adult heart is normalized to the transcript level in the skeletal muscle, set as 1. Bars represent the mean ± SD. * denotes P
    Figure Legend Snippet: Expression of the zebrafish MARP genes in adult heart and skeletal muscle. Quantification of ankrd1a , ankrd1b and ankrd2 expression was done by qPCR, using mRNA isolated from the hearts of 8 fish, pooled in 4 groups and the skeletal muscles of 4 fish. Relative level of transcripts in adult heart is normalized to the transcript level in the skeletal muscle, set as 1. Bars represent the mean ± SD. * denotes P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Isolation, Fluorescence In Situ Hybridization

    Quantification of zebrafish MARP transcripts during development. Expression levels of ankrd1a , ankrd1b and ankrd2 at indicated time points after fertilization were obtained using qPCR. The housekeeping gene rlp13a served as internal reference. Data (mean ± SD) are combined from four biological replicates and normalized to the 24 hpf time point. * denotes P
    Figure Legend Snippet: Quantification of zebrafish MARP transcripts during development. Expression levels of ankrd1a , ankrd1b and ankrd2 at indicated time points after fertilization were obtained using qPCR. The housekeeping gene rlp13a served as internal reference. Data (mean ± SD) are combined from four biological replicates and normalized to the 24 hpf time point. * denotes P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

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    Solis BioDyne hot firepol evagreen qpcr mix
    Intraperitoneal administration of ethidium bromide activates UPR mt in muscle. Balb/c mices were injected though the intraperitoneal route, with 2 different concentration of ethidium bromide (10 mg/kg; 50 mg/kg) and methacycline hydrochloride (100 mg/kg; 200 mg/kg). The quadriceps muscle from mouse hind limb was isolated after 16 hrs of drug administration. The muscle RNA was isolated and expression of mitochondrial specific chaperones and protease (hspd1, hsp10, hsp75 and clpp1) were measured using <t>QPCR.</t> The bars show the average ± SEM of 3 mice per group (***: p
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    Intraperitoneal administration of ethidium bromide activates UPR mt in muscle. Balb/c mices were injected though the intraperitoneal route, with 2 different concentration of ethidium bromide (10 mg/kg; 50 mg/kg) and methacycline hydrochloride (100 mg/kg; 200 mg/kg). The quadriceps muscle from mouse hind limb was isolated after 16 hrs of drug administration. The muscle RNA was isolated and expression of mitochondrial specific chaperones and protease (hspd1, hsp10, hsp75 and clpp1) were measured using QPCR. The bars show the average ± SEM of 3 mice per group (***: p

    Journal: Worm

    Article Title: A chemical screen to identify inducers of the mitochondrial unfolded protein response in C. elegans

    doi: 10.1080/21624054.2015.1096490

    Figure Lengend Snippet: Intraperitoneal administration of ethidium bromide activates UPR mt in muscle. Balb/c mices were injected though the intraperitoneal route, with 2 different concentration of ethidium bromide (10 mg/kg; 50 mg/kg) and methacycline hydrochloride (100 mg/kg; 200 mg/kg). The quadriceps muscle from mouse hind limb was isolated after 16 hrs of drug administration. The muscle RNA was isolated and expression of mitochondrial specific chaperones and protease (hspd1, hsp10, hsp75 and clpp1) were measured using QPCR. The bars show the average ± SEM of 3 mice per group (***: p

    Article Snippet: DNAse treatment of the RNA sample was also carried out to eliminate trace DNA contaminations. cDNA from the isolated muscle tissue RNA was then synthesized using an ABI high-capacity cDNA RT kit and the QPCR was performed using a 5 × HOT FIREPOL EvaGreen qPCR Mix; Cat no. 08-36-00008) from SOLIS BIODYNE.

    Techniques: Injection, Concentration Assay, Isolation, Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

    Biochemical and genetic characterization of mitochondrial phenotype of  mpv17  KO larvae.  (A) Basal OCR was measured for ∼1 h in fish water, immediately after 4 dpf larvae were exposed to 0.5 μM FCCP and, later, to a combination of 2 μM rotenone (Rot) and 5 μM antimycin (AA). Four independent experiments were performed ( n =40). (B) Quantification of basal respiration in wild-type and  mpv17 −/−  mutants ( n =40). (C) Protein blot analysis of different subunits of OXPHOS complexes. (D) Relative quantification of protein amount, using an antibody against βActin for standardization ( n =3). R.I., relative intensity. (E) Relative quantification of mtDNA copy number in wild type and  mpv17  homozygous mutants. Mean dCt values were calculated as Ct of  mt-nd1  (mitochondrially encoded gene) minus Ct of  polg  (nuclear gene) and plotted with s.e.m. ( n =7). (F) Real-time PCR quantification of mRNA transcripts from Ubiquinol-cytochrome c reductase complex subunits ( n =6). Statistical analyses were performed using two-tailed Student's  t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. **** P

    Journal: Disease Models & Mechanisms

    Article Title: The zebrafish orthologue of the human hepatocerebral disease gene MPV17 plays pleiotropic roles in mitochondria

    doi: 10.1242/dmm.037226

    Figure Lengend Snippet: Biochemical and genetic characterization of mitochondrial phenotype of mpv17 KO larvae. (A) Basal OCR was measured for ∼1 h in fish water, immediately after 4 dpf larvae were exposed to 0.5 μM FCCP and, later, to a combination of 2 μM rotenone (Rot) and 5 μM antimycin (AA). Four independent experiments were performed ( n =40). (B) Quantification of basal respiration in wild-type and mpv17 −/− mutants ( n =40). (C) Protein blot analysis of different subunits of OXPHOS complexes. (D) Relative quantification of protein amount, using an antibody against βActin for standardization ( n =3). R.I., relative intensity. (E) Relative quantification of mtDNA copy number in wild type and mpv17 homozygous mutants. Mean dCt values were calculated as Ct of mt-nd1 (mitochondrially encoded gene) minus Ct of polg (nuclear gene) and plotted with s.e.m. ( n =7). (F) Real-time PCR quantification of mRNA transcripts from Ubiquinol-cytochrome c reductase complex subunits ( n =6). Statistical analyses were performed using two-tailed Student's t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. **** P

    Article Snippet: Quantitative PCR (qPCR) was performed using a Rotor-gene Q (Qiagen) and 5× HOT FIREPol® EvaGreen® qPCR Mix Plus (Solis BioDyne), following the manufacturers’ protocols.

    Techniques: Fluorescence In Situ Hybridization, Real-time Polymerase Chain Reaction, Two Tailed Test

    Evaluation of stress response and mitochondrial quality control system in zebrafish larvae.  (A) Representative western blot analysis of Grp75 protein from 6 dpf zebrafish larvae and relative quantification, using an antibody against Tomm20 (also known as Tomm20b) for standardization ( n =3). (B) Real-time PCR quantification of mRNA transcripts from different MICOS subunits at 6 dpf ( n =8). Statistical analyses were performed using two-tailed Student's  t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. *** P

    Journal: Disease Models & Mechanisms

    Article Title: The zebrafish orthologue of the human hepatocerebral disease gene MPV17 plays pleiotropic roles in mitochondria

    doi: 10.1242/dmm.037226

    Figure Lengend Snippet: Evaluation of stress response and mitochondrial quality control system in zebrafish larvae. (A) Representative western blot analysis of Grp75 protein from 6 dpf zebrafish larvae and relative quantification, using an antibody against Tomm20 (also known as Tomm20b) for standardization ( n =3). (B) Real-time PCR quantification of mRNA transcripts from different MICOS subunits at 6 dpf ( n =8). Statistical analyses were performed using two-tailed Student's t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. *** P

    Article Snippet: Quantitative PCR (qPCR) was performed using a Rotor-gene Q (Qiagen) and 5× HOT FIREPol® EvaGreen® qPCR Mix Plus (Solis BioDyne), following the manufacturers’ protocols.

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

    Investigation of  mpv17  orthologue and paralogue genes in zebrafish larvae.  (A) Real-time PCR quantification of mRNA transcripts of  mpv17-like2  and  mpv17-like  at 6 dpf ( n =9). (B) Evaluation of phenotypic rescue in the tail region at 3 dpf after the injection of human  MPV17  mRNAs, wild-type (WT) and p.R50Q mutated forms, and zebrafish  mpv17  and  mpv17-like2  mRNAs. Arrowheads point to iridophores. Scale bars: 100 µm. (C) Relative quantification of iridophore amount in controls and injected larvae ( n =30). (D) mtDNA copy number analysis in  mpv17 −/−  mutants transiently overexpressing  mpv17-like2  at 3 dpf and 6 dpf. Mean dCt values were calculated as Ct of  mt-nd1  (mitochondrially encoded gene) minus Ct of  polg  (nuclear gene) ( n =4). Statistical analyses were performed using two-tailed Student's  t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. **** P

    Journal: Disease Models & Mechanisms

    Article Title: The zebrafish orthologue of the human hepatocerebral disease gene MPV17 plays pleiotropic roles in mitochondria

    doi: 10.1242/dmm.037226

    Figure Lengend Snippet: Investigation of mpv17 orthologue and paralogue genes in zebrafish larvae. (A) Real-time PCR quantification of mRNA transcripts of mpv17-like2 and mpv17-like at 6 dpf ( n =9). (B) Evaluation of phenotypic rescue in the tail region at 3 dpf after the injection of human MPV17 mRNAs, wild-type (WT) and p.R50Q mutated forms, and zebrafish mpv17 and mpv17-like2 mRNAs. Arrowheads point to iridophores. Scale bars: 100 µm. (C) Relative quantification of iridophore amount in controls and injected larvae ( n =30). (D) mtDNA copy number analysis in mpv17 −/− mutants transiently overexpressing mpv17-like2 at 3 dpf and 6 dpf. Mean dCt values were calculated as Ct of mt-nd1 (mitochondrially encoded gene) minus Ct of polg (nuclear gene) ( n =4). Statistical analyses were performed using two-tailed Student's t -test. Statistical significance was evaluated by setting a confidence interval of 95%; data are mean±s.e.m. **** P

    Article Snippet: Quantitative PCR (qPCR) was performed using a Rotor-gene Q (Qiagen) and 5× HOT FIREPol® EvaGreen® qPCR Mix Plus (Solis BioDyne), following the manufacturers’ protocols.

    Techniques: Real-time Polymerase Chain Reaction, Injection, Two Tailed Test

    Functional validation of PRO-cap enhancers. A) Comparing changes in eRNA expression with ENZ treatment as detected by PRO-cap with changes in H3K27Ac ChIP-seq and ATAC-seq. XY charts and heatmaps show read density 1kb up- and downstream of the center of the ENZ regulated enhancers. Data is from publicly available data 57 (GSE137775). B) Selected candidate enhancers for functional analysis. C) Analysis of changes in enhancer activity with androgen stimulation or inhibition as determined by luciferase reported assays does not agree with PRO-cap data. 24 hours post transfection cells were treated with 10 μM enzalutamide or 10 nM DHT for and additional 24 hours. Shown is the average log2 fold change between DMSO and ENZ or EtOH and DHT from n = 3 experiments, each with n = 3 replicates. Statistics were determined by t-test. D) Analysis of eRNA expression in response to AR stimulation (10 nM DHT) or inhibition (10 μM ENZ). Expression was measured via qPCR (n = 3) and is displayed as the average log2 fold change with treatment. Statistics were determined by t-test . E) Categorization of ENZ-regulated enhancers identified with PRO-cap in publish STARR-seq data 31 . Activity of 139 of the 853 candidate enhancers was measured in response to androgen stimulation (DHT) and categorized as inactive (no enhancer activity), constitutively active (no change in activity with DHT) or induced by DHT. Statistics were determined using Fisher’s exact test.

    Journal: bioRxiv

    Article Title: Capped nascent RNA sequencing reveals novel therapy-responsive enhancers in prostate cancer

    doi: 10.1101/2022.04.08.487666

    Figure Lengend Snippet: Functional validation of PRO-cap enhancers. A) Comparing changes in eRNA expression with ENZ treatment as detected by PRO-cap with changes in H3K27Ac ChIP-seq and ATAC-seq. XY charts and heatmaps show read density 1kb up- and downstream of the center of the ENZ regulated enhancers. Data is from publicly available data 57 (GSE137775). B) Selected candidate enhancers for functional analysis. C) Analysis of changes in enhancer activity with androgen stimulation or inhibition as determined by luciferase reported assays does not agree with PRO-cap data. 24 hours post transfection cells were treated with 10 μM enzalutamide or 10 nM DHT for and additional 24 hours. Shown is the average log2 fold change between DMSO and ENZ or EtOH and DHT from n = 3 experiments, each with n = 3 replicates. Statistics were determined by t-test. D) Analysis of eRNA expression in response to AR stimulation (10 nM DHT) or inhibition (10 μM ENZ). Expression was measured via qPCR (n = 3) and is displayed as the average log2 fold change with treatment. Statistics were determined by t-test . E) Categorization of ENZ-regulated enhancers identified with PRO-cap in publish STARR-seq data 31 . Activity of 139 of the 853 candidate enhancers was measured in response to androgen stimulation (DHT) and categorized as inactive (no enhancer activity), constitutively active (no change in activity with DHT) or induced by DHT. Statistics were determined using Fisher’s exact test.

    Article Snippet: Quantitative real-time PCR was performed on the ViiA 7 system (Applied Biosystems) using HOT FIREPol EvaGreen qPCR mix (Solis Biodyne, 08-24-00020) following manufacturer’s instruction.

    Techniques: Functional Assay, Expressing, Chromatin Immunoprecipitation, Activity Assay, Inhibition, Luciferase, Transfection, Real-time Polymerase Chain Reaction

    Identification of two point mutations in LAT gene of J.CaM2 cells by DNA sequencing and PCR–RFLP. ( a ) Left panel: histograms showing homozygous wild type 'C' nucleotide in intron 1 at the position g.237 in Jurkat and heterozygous variant C > T found in J.CaM2. The mutation was further confirmed by the digestion of PCR product (amplified from genomic DNA) with Bam HI endonuclease. Right panel: histogram of homozygous, wild type 'C' base in exon 4 at position c.167 in Jurkat and heterozygous c.167C > T missense mutation in J.CaM2 changing threonine to methionine at position 56. Presence of the variant was verified by the digestion of PCR product (generated using cDNA) with Nla III. ( b ) Scheme of the pGL4.14– LAT (−916/+357) plasmids used to test the effect of g.237C > T mutation on LAT promoter activity in Jurkat cells under resting and activation conditions. The results of dual-luciferase assay are representative of four technical replicates that represent two independent experiments ( P

    Journal: Genes and Immunity

    Article Title: Phorbol ester-mediated re-expression of endogenous LAT adapter in J.CaM2 cells: a model for dissecting drivers and blockers of LAT transcription

    doi: 10.1038/gene.2016.25

    Figure Lengend Snippet: Identification of two point mutations in LAT gene of J.CaM2 cells by DNA sequencing and PCR–RFLP. ( a ) Left panel: histograms showing homozygous wild type 'C' nucleotide in intron 1 at the position g.237 in Jurkat and heterozygous variant C > T found in J.CaM2. The mutation was further confirmed by the digestion of PCR product (amplified from genomic DNA) with Bam HI endonuclease. Right panel: histogram of homozygous, wild type 'C' base in exon 4 at position c.167 in Jurkat and heterozygous c.167C > T missense mutation in J.CaM2 changing threonine to methionine at position 56. Presence of the variant was verified by the digestion of PCR product (generated using cDNA) with Nla III. ( b ) Scheme of the pGL4.14– LAT (−916/+357) plasmids used to test the effect of g.237C > T mutation on LAT promoter activity in Jurkat cells under resting and activation conditions. The results of dual-luciferase assay are representative of four technical replicates that represent two independent experiments ( P

    Article Snippet: Amplifications were carried out with 5 × HOT FIREPol EvaGreen Quantitative PCR Mix Plus with ROX (Solis BioDyne, Tartu, Estonia).

    Techniques: DNA Sequencing, Polymerase Chain Reaction, Variant Assay, Mutagenesis, Amplification, Generated, Activity Assay, Activation Assay, Luciferase

    Histone modifications and recruitment of Sp transcription factors associated with the steady-state and PMA-induced expression of LAT. ( a ) ChIP-seq profiles derived from publicly available ChIP-seq data sets for RNA Polymerase II (Pol II; GSE50622), DNase I hypersensitive sites (DHS; GSE29692), H3K4me3 (GSE35583) and H3K27ac (GSE59257)—visualized for LAT gene in resting Jurkat cells. The region defined earlier using dual-reporter assay 10 as LAT proximal promoter (P) is bound by Pol II and displays DNase I hypersensitivity. The promoter is also occupied by H3K4me3 and H3K27ac that are indicative of active promoters. ( b ) Representative ChIP–PCR results revealing binding signals of H3K27ac, Sp1 and Sp3 on chosen LAT gene regions in untreated and PMA stimulated J.CaM2 cells. The experiment was repeated four times giving reproducible results.

    Journal: Genes and Immunity

    Article Title: Phorbol ester-mediated re-expression of endogenous LAT adapter in J.CaM2 cells: a model for dissecting drivers and blockers of LAT transcription

    doi: 10.1038/gene.2016.25

    Figure Lengend Snippet: Histone modifications and recruitment of Sp transcription factors associated with the steady-state and PMA-induced expression of LAT. ( a ) ChIP-seq profiles derived from publicly available ChIP-seq data sets for RNA Polymerase II (Pol II; GSE50622), DNase I hypersensitive sites (DHS; GSE29692), H3K4me3 (GSE35583) and H3K27ac (GSE59257)—visualized for LAT gene in resting Jurkat cells. The region defined earlier using dual-reporter assay 10 as LAT proximal promoter (P) is bound by Pol II and displays DNase I hypersensitivity. The promoter is also occupied by H3K4me3 and H3K27ac that are indicative of active promoters. ( b ) Representative ChIP–PCR results revealing binding signals of H3K27ac, Sp1 and Sp3 on chosen LAT gene regions in untreated and PMA stimulated J.CaM2 cells. The experiment was repeated four times giving reproducible results.

    Article Snippet: Amplifications were carried out with 5 × HOT FIREPol EvaGreen Quantitative PCR Mix Plus with ROX (Solis BioDyne, Tartu, Estonia).

    Techniques: Expressing, Chromatin Immunoprecipitation, Derivative Assay, Reporter Assay, Polymerase Chain Reaction, Binding Assay