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FOXO1 represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells <t>(qRT-PCR)</t> ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after siRNA treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.
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1) Product Images from "MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes"

Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02813

FOXO1 represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells (qRT-PCR) ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after siRNA treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.
Figure Legend Snippet: FOXO1 represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells (qRT-PCR) ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after siRNA treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.

Techniques Used: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Activation Assay, Plasmid Preparation, Cell Culture, Transduction, Staining, Flow Cytometry, Cytometry

Significant upregulation of miR-31 targets after knock-down of miR-31. (A) Schematic overview of the method to determine the fraction of putative miR-31 target mRNA molecules that contain at least one miR-31 bs in their 3′-UTR. Depicted is the coverage (black bars, middle row) of n exons and the 3′-UTR (upper row) containing the miR-31 bs (indicated in blue). The ratio is calculated from the median coverage of the exons and the coverage of the miR-31 bs in the 3′-UTR (bottom row). (B) 421 putative miR-31 targets were grouped according to the ratio determined in (A) . (C) MiR-31 expression in Th1 rep cells 24, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR normalized to snU6 determined by qRT-PCR. Data is shown as mean +SEM, n = 11, pooled from five independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). (D) GSEA with the PT- and PT 50 - gene-sets and the transcriptome data of Th1 rep cells 36, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR. Data is shown as enrichment curves with each putative target gene (PT, black; PT 50 , green) in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (E) Welch's test of nominal enrichment scores (NES) from 1,000 independent GSEA's each using a randomly chosen subset of PT with sizes equal to the size of the PT 50 set, p -value is depicted in the figure.
Figure Legend Snippet: Significant upregulation of miR-31 targets after knock-down of miR-31. (A) Schematic overview of the method to determine the fraction of putative miR-31 target mRNA molecules that contain at least one miR-31 bs in their 3′-UTR. Depicted is the coverage (black bars, middle row) of n exons and the 3′-UTR (upper row) containing the miR-31 bs (indicated in blue). The ratio is calculated from the median coverage of the exons and the coverage of the miR-31 bs in the 3′-UTR (bottom row). (B) 421 putative miR-31 targets were grouped according to the ratio determined in (A) . (C) MiR-31 expression in Th1 rep cells 24, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR normalized to snU6 determined by qRT-PCR. Data is shown as mean +SEM, n = 11, pooled from five independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). (D) GSEA with the PT- and PT 50 - gene-sets and the transcriptome data of Th1 rep cells 36, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR. Data is shown as enrichment curves with each putative target gene (PT, black; PT 50 , green) in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (E) Welch's test of nominal enrichment scores (NES) from 1,000 independent GSEA's each using a randomly chosen subset of PT with sizes equal to the size of the PT 50 set, p -value is depicted in the figure.

Techniques Used: Expressing, Activation Assay, Quantitative RT-PCR, MANN-WHITNEY

MiR-31 targets a set of genes involved in cytoskeletal rearrangement and miR-31 inhibition increases the motility of Th1 rep cells. (A) GSEA of the transcriptome data obtained from Th1 rep cells 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR with the KEGG-pathway database (v. 6.0) used as source for gene-sets. Data of two significantly enriched gene-sets is shown as enrichment curves with all genes in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (B) Network of validated functional interactions among positively correlated miR-31 targets (green rimmed; Figure 2D ) and the genes defining the gene set “regulation of actin cytoskeleton” (red). The resulting network was adapted to T cells (also see methods). Genes without interactions are not included. (C) QRT-PCR of target mRNA expression in reactivated Th1 rep cells 72 h after antagomir treatment relative to Hprt and normalized to Antagomir-SCR treated control. Data is shown as mean +SEM, n = 12 (for Lats2, RhoA, Stk40, Ywhae ) pooled from four independent experiments, or n = 6 (for Ablim1, Cd28, Cdc42, Eif4ebp2, LPP, Ppp2r2a, Ppp3ca, Rac1 ) pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, * p ≤ 0.05). (D,E) Transwell migration assays with an ICAM-1 coated membrane (10 μg/ml) and CXCL10 (100 ng/ml) in the lower compartment for once and repeatedly activated Th1 cells, 72 h after reactivation with αCD3/28 (D) and Th1 rep cells, 72 h after antagomir treatment and reactivation with αCD3/28 (E) , assessed by flow cytometry, normalized to inserted cell number. Data is shown as mean +SEM, n = 16–18 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001).
Figure Legend Snippet: MiR-31 targets a set of genes involved in cytoskeletal rearrangement and miR-31 inhibition increases the motility of Th1 rep cells. (A) GSEA of the transcriptome data obtained from Th1 rep cells 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR with the KEGG-pathway database (v. 6.0) used as source for gene-sets. Data of two significantly enriched gene-sets is shown as enrichment curves with all genes in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (B) Network of validated functional interactions among positively correlated miR-31 targets (green rimmed; Figure 2D ) and the genes defining the gene set “regulation of actin cytoskeleton” (red). The resulting network was adapted to T cells (also see methods). Genes without interactions are not included. (C) QRT-PCR of target mRNA expression in reactivated Th1 rep cells 72 h after antagomir treatment relative to Hprt and normalized to Antagomir-SCR treated control. Data is shown as mean +SEM, n = 12 (for Lats2, RhoA, Stk40, Ywhae ) pooled from four independent experiments, or n = 6 (for Ablim1, Cd28, Cdc42, Eif4ebp2, LPP, Ppp2r2a, Ppp3ca, Rac1 ) pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, * p ≤ 0.05). (D,E) Transwell migration assays with an ICAM-1 coated membrane (10 μg/ml) and CXCL10 (100 ng/ml) in the lower compartment for once and repeatedly activated Th1 cells, 72 h after reactivation with αCD3/28 (D) and Th1 rep cells, 72 h after antagomir treatment and reactivation with αCD3/28 (E) , assessed by flow cytometry, normalized to inserted cell number. Data is shown as mean +SEM, n = 16–18 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001).

Techniques Used: Inhibition, Activation Assay, Functional Assay, Quantitative RT-PCR, Expressing, MANN-WHITNEY, Migration, Flow Cytometry, Cytometry

TCR/CD28 induced expression of mir-31 is increased by T-Bet and IFN-γ. (A) MiR-31 expression kinetics normalized to snU6 after the first activation of naive CD4 + (left panel) or reactivation of Th1 rep cells (right panel) with αCD3/28, presented relative to values obtained from naive CD4 + cells ex vivo , determined by qRT-PCR. Data is shown as mean ±SEM, n = 8–12 pooled from 3 to 4 independent experiments (One-way Anova with Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (B) RNA-Seq coverage of the miR-31 gene locus in Th1 rep cells (upper row) and analysis of published ChIP-seq data from Th1 cells for p300 ( 28 ), H3K4me3 and H3K27me3 ( 29 ) using the Cistrome Browser (lower row). (C) Analysis of the putative promoter region as determined in (B) using published ChIP-Seq data obtained from Th1 cells for T-Bet ( 34 ), STAT1 ( 28 ), and STAT4 ( 33 ) and from naive CD4 + T cells ( 35 ), as well as predicted conserved binding sites for these transcription factors obtained from ECR Browser. (D) Schematic overview of the murine miR-31 gene locus and the resulting primary transcript as analyzed in (B) . (E) MiR-31 expression normalized to Hprt in naive CD4 + cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ± IFN-γ (10 ng/ml), presented relative to values obtained from naive CD4 + cells ex vivo determined by qRT-PCR. Data is shown as mean ±SEM, n = 4–8 pooled from two independent experiments (One-way Anova with Dunn's test for multiple comparison, ** p ≤ 0.01).
Figure Legend Snippet: TCR/CD28 induced expression of mir-31 is increased by T-Bet and IFN-γ. (A) MiR-31 expression kinetics normalized to snU6 after the first activation of naive CD4 + (left panel) or reactivation of Th1 rep cells (right panel) with αCD3/28, presented relative to values obtained from naive CD4 + cells ex vivo , determined by qRT-PCR. Data is shown as mean ±SEM, n = 8–12 pooled from 3 to 4 independent experiments (One-way Anova with Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (B) RNA-Seq coverage of the miR-31 gene locus in Th1 rep cells (upper row) and analysis of published ChIP-seq data from Th1 cells for p300 ( 28 ), H3K4me3 and H3K27me3 ( 29 ) using the Cistrome Browser (lower row). (C) Analysis of the putative promoter region as determined in (B) using published ChIP-Seq data obtained from Th1 cells for T-Bet ( 34 ), STAT1 ( 28 ), and STAT4 ( 33 ) and from naive CD4 + T cells ( 35 ), as well as predicted conserved binding sites for these transcription factors obtained from ECR Browser. (D) Schematic overview of the murine miR-31 gene locus and the resulting primary transcript as analyzed in (B) . (E) MiR-31 expression normalized to Hprt in naive CD4 + cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ± IFN-γ (10 ng/ml), presented relative to values obtained from naive CD4 + cells ex vivo determined by qRT-PCR. Data is shown as mean ±SEM, n = 4–8 pooled from two independent experiments (One-way Anova with Dunn's test for multiple comparison, ** p ≤ 0.01).

Techniques Used: Expressing, Activation Assay, Ex Vivo, Quantitative RT-PCR, MANN-WHITNEY, RNA Sequencing Assay, Chromatin Immunoprecipitation, Binding Assay

MiR-31 is upregulated in murine Th1 rep cells, and in memory Th cells from the synovial fluid of RA patients. (A) MiR-31 expression in once (day 6) and repeatedly (three rounds of restimulation with 6 day intervals) activated Th1, Th2, Th17, and ex vivo isolated naive CD4 + cells normalized to snU6 determined by qRT-PCR. Each data point represents an independent experiment ( n = 12 [naive and Th1], 5 [Th2], 4 [Th17]) (Wilcoxon-Test for paired data, *** p ≤ 0.001). (B) MiR-31 expression normalized to snU6 in CD3 + CD4 + CD14 − CD45RO + T cells isolated from the synovial fluid of patients suffering from RA or blood from healthy control (HC) donors ex vivo or after 3 h of restimulation with PMA/ionomycin (P/I) ( n = 5 RA; n = 4 HC) determined by qRT-PCR. Each data point represents an individual donor, horizontal bar: median (Mann-Whitney test for unpaired data, * p ≤ 0.05).
Figure Legend Snippet: MiR-31 is upregulated in murine Th1 rep cells, and in memory Th cells from the synovial fluid of RA patients. (A) MiR-31 expression in once (day 6) and repeatedly (three rounds of restimulation with 6 day intervals) activated Th1, Th2, Th17, and ex vivo isolated naive CD4 + cells normalized to snU6 determined by qRT-PCR. Each data point represents an independent experiment ( n = 12 [naive and Th1], 5 [Th2], 4 [Th17]) (Wilcoxon-Test for paired data, *** p ≤ 0.001). (B) MiR-31 expression normalized to snU6 in CD3 + CD4 + CD14 − CD45RO + T cells isolated from the synovial fluid of patients suffering from RA or blood from healthy control (HC) donors ex vivo or after 3 h of restimulation with PMA/ionomycin (P/I) ( n = 5 RA; n = 4 HC) determined by qRT-PCR. Each data point represents an individual donor, horizontal bar: median (Mann-Whitney test for unpaired data, * p ≤ 0.05).

Techniques Used: Expressing, Ex Vivo, Isolation, Quantitative RT-PCR, MANN-WHITNEY

2) Product Images from "MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes"

Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02813

FOXO1 represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells (qRT-PCR) ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after siRNA treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.
Figure Legend Snippet: FOXO1 represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells (qRT-PCR) ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after siRNA treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.

Techniques Used: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Activation Assay, Plasmid Preparation, Cell Culture, Transduction, Staining, Flow Cytometry, Cytometry

Significant upregulation of miR-31 targets after knock-down of miR-31. (A) Schematic overview of the method to determine the fraction of putative miR-31 target mRNA molecules that contain at least one miR-31 bs in their 3′-UTR. Depicted is the coverage (black bars, middle row) of n exons and the 3′-UTR (upper row) containing the miR-31 bs (indicated in blue). The ratio is calculated from the median coverage of the exons and the coverage of the miR-31 bs in the 3′-UTR (bottom row). (B) 421 putative miR-31 targets were grouped according to the ratio determined in (A) . (C) MiR-31 expression in Th1 rep cells 24, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR normalized to snU6 determined by qRT-PCR. Data is shown as mean +SEM, n = 11, pooled from five independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). (D) GSEA with the PT- and PT 50 - gene-sets and the transcriptome data of Th1 rep cells 36, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR. Data is shown as enrichment curves with each putative target gene (PT, black; PT 50 , green) in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (E) Welch's test of nominal enrichment scores (NES) from 1,000 independent GSEA's each using a randomly chosen subset of PT with sizes equal to the size of the PT 50 set, p -value is depicted in the figure.
Figure Legend Snippet: Significant upregulation of miR-31 targets after knock-down of miR-31. (A) Schematic overview of the method to determine the fraction of putative miR-31 target mRNA molecules that contain at least one miR-31 bs in their 3′-UTR. Depicted is the coverage (black bars, middle row) of n exons and the 3′-UTR (upper row) containing the miR-31 bs (indicated in blue). The ratio is calculated from the median coverage of the exons and the coverage of the miR-31 bs in the 3′-UTR (bottom row). (B) 421 putative miR-31 targets were grouped according to the ratio determined in (A) . (C) MiR-31 expression in Th1 rep cells 24, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR normalized to snU6 determined by qRT-PCR. Data is shown as mean +SEM, n = 11, pooled from five independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). (D) GSEA with the PT- and PT 50 - gene-sets and the transcriptome data of Th1 rep cells 36, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR. Data is shown as enrichment curves with each putative target gene (PT, black; PT 50 , green) in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (E) Welch's test of nominal enrichment scores (NES) from 1,000 independent GSEA's each using a randomly chosen subset of PT with sizes equal to the size of the PT 50 set, p -value is depicted in the figure.

Techniques Used: Expressing, Activation Assay, Quantitative RT-PCR, MANN-WHITNEY

TCR/CD28 induced expression of mir-31 is increased by T-Bet and IFN-γ. (A) MiR-31 expression kinetics normalized to snU6 after the first activation of naive CD4 + (left panel) or reactivation of Th1 rep cells (right panel) with αCD3/28, presented relative to values obtained from naive CD4 + cells ex vivo , determined by qRT-PCR. Data is shown as mean ±SEM, n = 8–12 pooled from 3 to 4 independent experiments (One-way Anova with Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (B) ) using the Cistrome Browser (lower row). (C) Analysis of the putative promoter region as determined in (B) ) and from naive CD4 + ), as well as predicted conserved binding sites for these transcription factors obtained from ECR Browser. (D) Schematic overview of the murine miR-31 gene locus and the resulting primary transcript as analyzed in (B) . (E) MiR-31 expression normalized to Hprt in naive CD4 + cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ± IFN-γ (10 ng/ml), presented relative to values obtained from naive CD4 + cells ex vivo determined by qRT-PCR. Data is shown as mean ±SEM, n = 4–8 pooled from two independent experiments (One-way Anova with Dunn's test for multiple comparison, ** p ≤ 0.01).
Figure Legend Snippet: TCR/CD28 induced expression of mir-31 is increased by T-Bet and IFN-γ. (A) MiR-31 expression kinetics normalized to snU6 after the first activation of naive CD4 + (left panel) or reactivation of Th1 rep cells (right panel) with αCD3/28, presented relative to values obtained from naive CD4 + cells ex vivo , determined by qRT-PCR. Data is shown as mean ±SEM, n = 8–12 pooled from 3 to 4 independent experiments (One-way Anova with Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (B) ) using the Cistrome Browser (lower row). (C) Analysis of the putative promoter region as determined in (B) ) and from naive CD4 + ), as well as predicted conserved binding sites for these transcription factors obtained from ECR Browser. (D) Schematic overview of the murine miR-31 gene locus and the resulting primary transcript as analyzed in (B) . (E) MiR-31 expression normalized to Hprt in naive CD4 + cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ± IFN-γ (10 ng/ml), presented relative to values obtained from naive CD4 + cells ex vivo determined by qRT-PCR. Data is shown as mean ±SEM, n = 4–8 pooled from two independent experiments (One-way Anova with Dunn's test for multiple comparison, ** p ≤ 0.01).

Techniques Used: Expressing, Activation Assay, Ex Vivo, Quantitative RT-PCR, MANN-WHITNEY, Binding Assay

MiR-31 is upregulated in murine Th1 rep cells, and in memory Th cells from the synovial fluid of RA patients. (A) MiR-31 expression in once (day 6) and repeatedly (three rounds of restimulation with 6 day intervals) activated Th1, Th2, Th17, and ex vivo isolated naive CD4 + cells normalized to snU6 determined by qRT-PCR. Each data point represents an independent experiment ( n = 12 [naive and Th1], 5 [Th2], 4 [Th17]) (Wilcoxon-Test for paired data, *** p ≤ 0.001). (B) MiR-31 expression normalized to snU6 in CD3 + CD4 + CD14 − CD45RO + T cells isolated from the synovial fluid of patients suffering from RA or blood from healthy control (HC) donors ex vivo or after 3 h of restimulation with PMA/ionomycin (P/I) ( n = 5 RA; n = 4 HC) determined by qRT-PCR. Each data point represents an individual donor, horizontal bar: median (Mann-Whitney test for unpaired data, * p ≤ 0.05).
Figure Legend Snippet: MiR-31 is upregulated in murine Th1 rep cells, and in memory Th cells from the synovial fluid of RA patients. (A) MiR-31 expression in once (day 6) and repeatedly (three rounds of restimulation with 6 day intervals) activated Th1, Th2, Th17, and ex vivo isolated naive CD4 + cells normalized to snU6 determined by qRT-PCR. Each data point represents an independent experiment ( n = 12 [naive and Th1], 5 [Th2], 4 [Th17]) (Wilcoxon-Test for paired data, *** p ≤ 0.001). (B) MiR-31 expression normalized to snU6 in CD3 + CD4 + CD14 − CD45RO + T cells isolated from the synovial fluid of patients suffering from RA or blood from healthy control (HC) donors ex vivo or after 3 h of restimulation with PMA/ionomycin (P/I) ( n = 5 RA; n = 4 HC) determined by qRT-PCR. Each data point represents an individual donor, horizontal bar: median (Mann-Whitney test for unpaired data, * p ≤ 0.05).

Techniques Used: Expressing, Ex Vivo, Isolation, Quantitative RT-PCR, MANN-WHITNEY

Related Articles

SYBR Green Assay:

Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes
Article Snippet: .. Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′. .. Alternatively mRNA was quantified by qRT-PCR based TaqMan Assays (ThermoFisher) using the following assays: ABLIM1 Mm01254316_m1, CD28 Mm01253994_m1, CD69 Mm01183378_m1, CDC42 Mm01194005_g1, EIF4EBP2 Mm00515675_m1, FOXO1 Mm00490671_m1, FOXO3 Mm01185722_m1, HPRT Mm03024075_m1, INFG Mm01168134_m1, KLF2 Mm01244979_g1, LATS2 Mm00497217_m1, LPP Mm00724478_m1, PPP2R2A Mm01317426_g1, PPP3CA Mm01317678_m1, Pri-miR-31 Mm03306874, RAC1 Mm01201653_mH, RHOA Mm00834507_g1, SELL Mm00441291_m1, STK40 Mm00512134_m1, and YWHAE Mm00494242_m1.

Article Title: PIP5K1α promotes myogenic differentiation via AKT activation and calcium release
Article Snippet: .. Following the manufacturer’s instructions, the expression level of PIP5K1 was detected by SYBR Green-based qRT-PCR with FastStart Universal SYBR Green Master mix (Roche). ..

Reverse Transcription Polymerase Chain Reaction:

Article Title: Overexpression of CXCR4 is significantly associated with cisplatin-based chemotherapy resistance and can be a prognostic factor in epithelial ovarian cancer
Article Snippet: Reverse transcription was performed using PrimeScript RT-PCR Kit (Takara). .. CXCR4 and GAPDH expression were analyzed by SYBR GREEN-based qRT- PCR (LightCycler480 system, Roche, WI, USA).

Isolation:

Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes
Article Snippet: Paragraph title: RNA Isolation and qRT-PCR ... Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′.

Quantitative RT-PCR:

Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes
Article Snippet: .. Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′. .. Alternatively mRNA was quantified by qRT-PCR based TaqMan Assays (ThermoFisher) using the following assays: ABLIM1 Mm01254316_m1, CD28 Mm01253994_m1, CD69 Mm01183378_m1, CDC42 Mm01194005_g1, EIF4EBP2 Mm00515675_m1, FOXO1 Mm00490671_m1, FOXO3 Mm01185722_m1, HPRT Mm03024075_m1, INFG Mm01168134_m1, KLF2 Mm01244979_g1, LATS2 Mm00497217_m1, LPP Mm00724478_m1, PPP2R2A Mm01317426_g1, PPP3CA Mm01317678_m1, Pri-miR-31 Mm03306874, RAC1 Mm01201653_mH, RHOA Mm00834507_g1, SELL Mm00441291_m1, STK40 Mm00512134_m1, and YWHAE Mm00494242_m1.

Article Title: Overexpression of CXCR4 is significantly associated with cisplatin-based chemotherapy resistance and can be a prognostic factor in epithelial ovarian cancer
Article Snippet: .. CXCR4 and GAPDH expression were analyzed by SYBR GREEN-based qRT- PCR (LightCycler480 system, Roche, WI, USA). ..

Article Title: PIP5K1α promotes myogenic differentiation via AKT activation and calcium release
Article Snippet: .. Following the manufacturer’s instructions, the expression level of PIP5K1 was detected by SYBR Green-based qRT-PCR with FastStart Universal SYBR Green Master mix (Roche). ..

Article Title: Control of embryonic stem cell self-renewal and differentiation via coordinated alternative splicing and translation of YY2
Article Snippet: .. Quantification of ChIP enrichment was carried out with SYBR Green-based qRT-PCR (04887352001; Roche) and the Roche LightCycler instrument. .. Sequencing libraries of ChIP DNA and corresponding 10% (wt/wt) chromatin inputs were prepared at the McGill University and Génome Québec Innovation Centre, following quality assessment with the Agilent Bioanalyzer 2100.

Purification:

Article Title: Control of embryonic stem cell self-renewal and differentiation via coordinated alternative splicing and translation of YY2
Article Snippet: DNA was purified with the Qiagen PCR Purification Kit (28106; Qiagen). .. Quantification of ChIP enrichment was carried out with SYBR Green-based qRT-PCR (04887352001; Roche) and the Roche LightCycler instrument.

Real-time Polymerase Chain Reaction:

Article Title: PIP5K1α promotes myogenic differentiation via AKT activation and calcium release
Article Snippet: Paragraph title: RNA preparation and quantitative real-time PCR ... Following the manufacturer’s instructions, the expression level of PIP5K1 was detected by SYBR Green-based qRT-PCR with FastStart Universal SYBR Green Master mix (Roche).

Polymerase Chain Reaction:

Article Title: Control of embryonic stem cell self-renewal and differentiation via coordinated alternative splicing and translation of YY2
Article Snippet: DNA was purified with the Qiagen PCR Purification Kit (28106; Qiagen). .. Quantification of ChIP enrichment was carried out with SYBR Green-based qRT-PCR (04887352001; Roche) and the Roche LightCycler instrument.

Expressing:

Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes
Article Snippet: .. Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′. .. Alternatively mRNA was quantified by qRT-PCR based TaqMan Assays (ThermoFisher) using the following assays: ABLIM1 Mm01254316_m1, CD28 Mm01253994_m1, CD69 Mm01183378_m1, CDC42 Mm01194005_g1, EIF4EBP2 Mm00515675_m1, FOXO1 Mm00490671_m1, FOXO3 Mm01185722_m1, HPRT Mm03024075_m1, INFG Mm01168134_m1, KLF2 Mm01244979_g1, LATS2 Mm00497217_m1, LPP Mm00724478_m1, PPP2R2A Mm01317426_g1, PPP3CA Mm01317678_m1, Pri-miR-31 Mm03306874, RAC1 Mm01201653_mH, RHOA Mm00834507_g1, SELL Mm00441291_m1, STK40 Mm00512134_m1, and YWHAE Mm00494242_m1.

Article Title: Overexpression of CXCR4 is significantly associated with cisplatin-based chemotherapy resistance and can be a prognostic factor in epithelial ovarian cancer
Article Snippet: .. CXCR4 and GAPDH expression were analyzed by SYBR GREEN-based qRT- PCR (LightCycler480 system, Roche, WI, USA). ..

Article Title: PIP5K1α promotes myogenic differentiation via AKT activation and calcium release
Article Snippet: .. Following the manufacturer’s instructions, the expression level of PIP5K1 was detected by SYBR Green-based qRT-PCR with FastStart Universal SYBR Green Master mix (Roche). ..

Sequencing:

Article Title: Control of embryonic stem cell self-renewal and differentiation via coordinated alternative splicing and translation of YY2
Article Snippet: Quantification of ChIP enrichment was carried out with SYBR Green-based qRT-PCR (04887352001; Roche) and the Roche LightCycler instrument. .. Sequencing libraries of ChIP DNA and corresponding 10% (wt/wt) chromatin inputs were prepared at the McGill University and Génome Québec Innovation Centre, following quality assessment with the Agilent Bioanalyzer 2100.

Chromatin Immunoprecipitation:

Article Title: Control of embryonic stem cell self-renewal and differentiation via coordinated alternative splicing and translation of YY2
Article Snippet: .. Quantification of ChIP enrichment was carried out with SYBR Green-based qRT-PCR (04887352001; Roche) and the Roche LightCycler instrument. .. Sequencing libraries of ChIP DNA and corresponding 10% (wt/wt) chromatin inputs were prepared at the McGill University and Génome Québec Innovation Centre, following quality assessment with the Agilent Bioanalyzer 2100.

ChIP-sequencing:

Article Title: Control of embryonic stem cell self-renewal and differentiation via coordinated alternative splicing and translation of YY2
Article Snippet: Paragraph title: ChIP and ChIP-Seq Assays. ... Quantification of ChIP enrichment was carried out with SYBR Green-based qRT-PCR (04887352001; Roche) and the Roche LightCycler instrument.

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    Roche sybr green mix based qrt pcr
    Treg secretion and function in both AE-WT and AE- fgl2 -/- mice after E . multilocularis infection, and in WT mice in response to recombinant FGL2/anti-FGL2-MAb blockade. (A) Spleen was taken from non-infected WT and AE-WT mice, CD4 + Teffs, CD8 + T cells, CD4 + CD25 + Tregs and APCs were isolated by FACS cell sorting, fgl2 mRNA levels were determined by <t>qRT-PCR.</t> (B) Representative images of Foxp3 mean fluorescence intensity (MFI) from AE-WT and AE- fgl2 -/- mice, and non-infected mice as controls. (C) Frequency of Foxp3 + T cells within CD4 + CD25 + T cells in PECs and spleen cells from AE-WT and AE- fgl2 -/- mice at 1 month and 4 months post-infection. (D) Foxp3 and IL-10 gene expression in PECs during E . multilocularis infection (measured by qRT-PCR). AU: arbitrary units. Graphs show the mean±SD. Data represent mean±SD of three independent experiments of a total 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA for statistical analysis. (E) 0, 1, and 5 μg/mL of recombinant FGL2 and 1μg/mL of anti-FGL2-MAb were added to primary spleen cells from non-infected WT mice, or spleen cells stimulated with ConA or vesicle fluid (VF). Relative expression levels of Foxp3 + /CD4 + CD25 + were determined by flow cytometry. (F) CD4 + CD25 + Tregs (suppressor cells) and CD4 + CD25 - Teff cells (responder cells) were isolated from spleen cells of both non-infected and infected AE-WT mice by FACS. The two cell populations were co-cultured at a ratio of 1:1 (suppressor: responder) in the presence of APCs and ConA (2 μg/mL), rFGL2 (1 μg/mL), ConA (2 μg/mL) + rFGL2 (1 μg/mL), or anti-FGL2-MAb (1 μg/mL); cell proliferation was measured using BrdU ELISA. Data represent mean±SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Expression of Foxp3 + /CD4+CD25 + was normalized with negative control (cells without rFGL2 and anti-FGL2-MAb (negative control) were considered as base line, e.g. as 1.0). Comparison between groups was performed using a one-way ANOVA with Bonferroni’s multiple comparison post-test for statistical analysis. * P
    Sybr Green Mix Based Qrt Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche sybr green quantitative real time polymerase chain reaction qrt pcr
    Expression of NF-κB1 and Bcl-2 detection in glioma tissues by <t>qRT-PCR.</t> Notes: The expression of NF-κB1 in glioma was significantly higher than that in nonneoplastic brain tissues ( P
    Sybr Green Quantitative Real Time Polymerase Chain Reaction Qrt Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Treg secretion and function in both AE-WT and AE- fgl2 -/- mice after E . multilocularis infection, and in WT mice in response to recombinant FGL2/anti-FGL2-MAb blockade. (A) Spleen was taken from non-infected WT and AE-WT mice, CD4 + Teffs, CD8 + T cells, CD4 + CD25 + Tregs and APCs were isolated by FACS cell sorting, fgl2 mRNA levels were determined by qRT-PCR. (B) Representative images of Foxp3 mean fluorescence intensity (MFI) from AE-WT and AE- fgl2 -/- mice, and non-infected mice as controls. (C) Frequency of Foxp3 + T cells within CD4 + CD25 + T cells in PECs and spleen cells from AE-WT and AE- fgl2 -/- mice at 1 month and 4 months post-infection. (D) Foxp3 and IL-10 gene expression in PECs during E . multilocularis infection (measured by qRT-PCR). AU: arbitrary units. Graphs show the mean±SD. Data represent mean±SD of three independent experiments of a total 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA for statistical analysis. (E) 0, 1, and 5 μg/mL of recombinant FGL2 and 1μg/mL of anti-FGL2-MAb were added to primary spleen cells from non-infected WT mice, or spleen cells stimulated with ConA or vesicle fluid (VF). Relative expression levels of Foxp3 + /CD4 + CD25 + were determined by flow cytometry. (F) CD4 + CD25 + Tregs (suppressor cells) and CD4 + CD25 - Teff cells (responder cells) were isolated from spleen cells of both non-infected and infected AE-WT mice by FACS. The two cell populations were co-cultured at a ratio of 1:1 (suppressor: responder) in the presence of APCs and ConA (2 μg/mL), rFGL2 (1 μg/mL), ConA (2 μg/mL) + rFGL2 (1 μg/mL), or anti-FGL2-MAb (1 μg/mL); cell proliferation was measured using BrdU ELISA. Data represent mean±SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Expression of Foxp3 + /CD4+CD25 + was normalized with negative control (cells without rFGL2 and anti-FGL2-MAb (negative control) were considered as base line, e.g. as 1.0). Comparison between groups was performed using a one-way ANOVA with Bonferroni’s multiple comparison post-test for statistical analysis. * P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Deletion of Fibrinogen-like Protein 2 (FGL-2), a Novel CD4+ CD25+ Treg Effector Molecule, Leads to Improved Control of Echinococcus multilocularis Infection in Mice

    doi: 10.1371/journal.pntd.0003755

    Figure Lengend Snippet: Treg secretion and function in both AE-WT and AE- fgl2 -/- mice after E . multilocularis infection, and in WT mice in response to recombinant FGL2/anti-FGL2-MAb blockade. (A) Spleen was taken from non-infected WT and AE-WT mice, CD4 + Teffs, CD8 + T cells, CD4 + CD25 + Tregs and APCs were isolated by FACS cell sorting, fgl2 mRNA levels were determined by qRT-PCR. (B) Representative images of Foxp3 mean fluorescence intensity (MFI) from AE-WT and AE- fgl2 -/- mice, and non-infected mice as controls. (C) Frequency of Foxp3 + T cells within CD4 + CD25 + T cells in PECs and spleen cells from AE-WT and AE- fgl2 -/- mice at 1 month and 4 months post-infection. (D) Foxp3 and IL-10 gene expression in PECs during E . multilocularis infection (measured by qRT-PCR). AU: arbitrary units. Graphs show the mean±SD. Data represent mean±SD of three independent experiments of a total 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA for statistical analysis. (E) 0, 1, and 5 μg/mL of recombinant FGL2 and 1μg/mL of anti-FGL2-MAb were added to primary spleen cells from non-infected WT mice, or spleen cells stimulated with ConA or vesicle fluid (VF). Relative expression levels of Foxp3 + /CD4 + CD25 + were determined by flow cytometry. (F) CD4 + CD25 + Tregs (suppressor cells) and CD4 + CD25 - Teff cells (responder cells) were isolated from spleen cells of both non-infected and infected AE-WT mice by FACS. The two cell populations were co-cultured at a ratio of 1:1 (suppressor: responder) in the presence of APCs and ConA (2 μg/mL), rFGL2 (1 μg/mL), ConA (2 μg/mL) + rFGL2 (1 μg/mL), or anti-FGL2-MAb (1 μg/mL); cell proliferation was measured using BrdU ELISA. Data represent mean±SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Expression of Foxp3 + /CD4+CD25 + was normalized with negative control (cells without rFGL2 and anti-FGL2-MAb (negative control) were considered as base line, e.g. as 1.0). Comparison between groups was performed using a one-way ANOVA with Bonferroni’s multiple comparison post-test for statistical analysis. * P

    Article Snippet: SYBR-Green Mix-based qRT-PCR was carried out on a Rotor-Gene 6000 qPCR detection system (Corbett) with the FastStart Essential DNA Green Master (Roche, Basel, Switzerland) following the manufacturer’s instructions.

    Techniques: Mouse Assay, Infection, Recombinant, Isolation, FACS, Quantitative RT-PCR, Fluorescence, Expressing, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Negative Control

    Serum FGL2 levels in E . multilocularis infected mice, effect of IL-17A on FGL2 secretion in spleen cells, and effects of FGL2 on parasite load and proliferation in E . multilocularis infected mice. (A) Serum levels of FGL2 in infected AE-WT mice at different stages of infection, as compared to non-infected control mice (Control). (B) Different concentrations of recombinant IL-17A (0, 0.5, 1, 2 μg/mL) or anti-IL-17A MAbs (1 μg/ml) were added to primary spleen cells isolated from non-infected WT mice (4 months p.i.). FGL2-levels in culture supernatants were determined by ELISA. (C) Parasite load in AE-WT and AE- fgl2 -/- mice assessed by wet weight measurement at 1 month and 4 months post- infection. (D) Representative images of E . multilocularis infection in AE-WT and AE- fgl2 -/- mice at 4 months p.i.; arrows point at intraperitoneal metacestode tissue/lesions. (E) Parasite cell proliferation in AE-WT versus AE- fgl2 -/- assessed by em14-3-3 qRT-PCR at 4 months p.i, upon comparison to the constitutively expressed house-keeping Em II/3-10 gene. Data represent mean ± SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA for statistical analysis. * P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Deletion of Fibrinogen-like Protein 2 (FGL-2), a Novel CD4+ CD25+ Treg Effector Molecule, Leads to Improved Control of Echinococcus multilocularis Infection in Mice

    doi: 10.1371/journal.pntd.0003755

    Figure Lengend Snippet: Serum FGL2 levels in E . multilocularis infected mice, effect of IL-17A on FGL2 secretion in spleen cells, and effects of FGL2 on parasite load and proliferation in E . multilocularis infected mice. (A) Serum levels of FGL2 in infected AE-WT mice at different stages of infection, as compared to non-infected control mice (Control). (B) Different concentrations of recombinant IL-17A (0, 0.5, 1, 2 μg/mL) or anti-IL-17A MAbs (1 μg/ml) were added to primary spleen cells isolated from non-infected WT mice (4 months p.i.). FGL2-levels in culture supernatants were determined by ELISA. (C) Parasite load in AE-WT and AE- fgl2 -/- mice assessed by wet weight measurement at 1 month and 4 months post- infection. (D) Representative images of E . multilocularis infection in AE-WT and AE- fgl2 -/- mice at 4 months p.i.; arrows point at intraperitoneal metacestode tissue/lesions. (E) Parasite cell proliferation in AE-WT versus AE- fgl2 -/- assessed by em14-3-3 qRT-PCR at 4 months p.i, upon comparison to the constitutively expressed house-keeping Em II/3-10 gene. Data represent mean ± SD of three independent experiments of a total of 15–18 mice in each group (5–6 mice per group in each independent experiment). Comparison between groups was performed using a one-way ANOVA for statistical analysis. * P

    Article Snippet: SYBR-Green Mix-based qRT-PCR was carried out on a Rotor-Gene 6000 qPCR detection system (Corbett) with the FastStart Essential DNA Green Master (Roche, Basel, Switzerland) following the manufacturer’s instructions.

    Techniques: Infection, Mouse Assay, Recombinant, Isolation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    FOXO1 represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells (qRT-PCR) ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after siRNA treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.

    Journal: Frontiers in Immunology

    Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

    doi: 10.3389/fimmu.2018.02813

    Figure Lengend Snippet: FOXO1 represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells (qRT-PCR) ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after siRNA treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.

    Article Snippet: Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′.

    Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Activation Assay, Plasmid Preparation, Cell Culture, Transduction, Staining, Flow Cytometry, Cytometry

    Significant upregulation of miR-31 targets after knock-down of miR-31. (A) Schematic overview of the method to determine the fraction of putative miR-31 target mRNA molecules that contain at least one miR-31 bs in their 3′-UTR. Depicted is the coverage (black bars, middle row) of n exons and the 3′-UTR (upper row) containing the miR-31 bs (indicated in blue). The ratio is calculated from the median coverage of the exons and the coverage of the miR-31 bs in the 3′-UTR (bottom row). (B) 421 putative miR-31 targets were grouped according to the ratio determined in (A) . (C) MiR-31 expression in Th1 rep cells 24, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR normalized to snU6 determined by qRT-PCR. Data is shown as mean +SEM, n = 11, pooled from five independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). (D) GSEA with the PT- and PT 50 - gene-sets and the transcriptome data of Th1 rep cells 36, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR. Data is shown as enrichment curves with each putative target gene (PT, black; PT 50 , green) in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (E) Welch's test of nominal enrichment scores (NES) from 1,000 independent GSEA's each using a randomly chosen subset of PT with sizes equal to the size of the PT 50 set, p -value is depicted in the figure.

    Journal: Frontiers in Immunology

    Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

    doi: 10.3389/fimmu.2018.02813

    Figure Lengend Snippet: Significant upregulation of miR-31 targets after knock-down of miR-31. (A) Schematic overview of the method to determine the fraction of putative miR-31 target mRNA molecules that contain at least one miR-31 bs in their 3′-UTR. Depicted is the coverage (black bars, middle row) of n exons and the 3′-UTR (upper row) containing the miR-31 bs (indicated in blue). The ratio is calculated from the median coverage of the exons and the coverage of the miR-31 bs in the 3′-UTR (bottom row). (B) 421 putative miR-31 targets were grouped according to the ratio determined in (A) . (C) MiR-31 expression in Th1 rep cells 24, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR normalized to snU6 determined by qRT-PCR. Data is shown as mean +SEM, n = 11, pooled from five independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). (D) GSEA with the PT- and PT 50 - gene-sets and the transcriptome data of Th1 rep cells 36, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR. Data is shown as enrichment curves with each putative target gene (PT, black; PT 50 , green) in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (E) Welch's test of nominal enrichment scores (NES) from 1,000 independent GSEA's each using a randomly chosen subset of PT with sizes equal to the size of the PT 50 set, p -value is depicted in the figure.

    Article Snippet: Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′.

    Techniques: Expressing, Activation Assay, Quantitative RT-PCR, MANN-WHITNEY

    MiR-31 targets a set of genes involved in cytoskeletal rearrangement and miR-31 inhibition increases the motility of Th1 rep cells. (A) GSEA of the transcriptome data obtained from Th1 rep cells 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR with the KEGG-pathway database (v. 6.0) used as source for gene-sets. Data of two significantly enriched gene-sets is shown as enrichment curves with all genes in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (B) Network of validated functional interactions among positively correlated miR-31 targets (green rimmed; Figure 2D ) and the genes defining the gene set “regulation of actin cytoskeleton” (red). The resulting network was adapted to T cells (also see methods). Genes without interactions are not included. (C) QRT-PCR of target mRNA expression in reactivated Th1 rep cells 72 h after antagomir treatment relative to Hprt and normalized to Antagomir-SCR treated control. Data is shown as mean +SEM, n = 12 (for Lats2, RhoA, Stk40, Ywhae ) pooled from four independent experiments, or n = 6 (for Ablim1, Cd28, Cdc42, Eif4ebp2, LPP, Ppp2r2a, Ppp3ca, Rac1 ) pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, * p ≤ 0.05). (D,E) Transwell migration assays with an ICAM-1 coated membrane (10 μg/ml) and CXCL10 (100 ng/ml) in the lower compartment for once and repeatedly activated Th1 cells, 72 h after reactivation with αCD3/28 (D) and Th1 rep cells, 72 h after antagomir treatment and reactivation with αCD3/28 (E) , assessed by flow cytometry, normalized to inserted cell number. Data is shown as mean +SEM, n = 16–18 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001).

    Journal: Frontiers in Immunology

    Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

    doi: 10.3389/fimmu.2018.02813

    Figure Lengend Snippet: MiR-31 targets a set of genes involved in cytoskeletal rearrangement and miR-31 inhibition increases the motility of Th1 rep cells. (A) GSEA of the transcriptome data obtained from Th1 rep cells 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR with the KEGG-pathway database (v. 6.0) used as source for gene-sets. Data of two significantly enriched gene-sets is shown as enrichment curves with all genes in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (B) Network of validated functional interactions among positively correlated miR-31 targets (green rimmed; Figure 2D ) and the genes defining the gene set “regulation of actin cytoskeleton” (red). The resulting network was adapted to T cells (also see methods). Genes without interactions are not included. (C) QRT-PCR of target mRNA expression in reactivated Th1 rep cells 72 h after antagomir treatment relative to Hprt and normalized to Antagomir-SCR treated control. Data is shown as mean +SEM, n = 12 (for Lats2, RhoA, Stk40, Ywhae ) pooled from four independent experiments, or n = 6 (for Ablim1, Cd28, Cdc42, Eif4ebp2, LPP, Ppp2r2a, Ppp3ca, Rac1 ) pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, * p ≤ 0.05). (D,E) Transwell migration assays with an ICAM-1 coated membrane (10 μg/ml) and CXCL10 (100 ng/ml) in the lower compartment for once and repeatedly activated Th1 cells, 72 h after reactivation with αCD3/28 (D) and Th1 rep cells, 72 h after antagomir treatment and reactivation with αCD3/28 (E) , assessed by flow cytometry, normalized to inserted cell number. Data is shown as mean +SEM, n = 16–18 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001).

    Article Snippet: Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′.

    Techniques: Inhibition, Activation Assay, Functional Assay, Quantitative RT-PCR, Expressing, MANN-WHITNEY, Migration, Flow Cytometry, Cytometry

    TCR/CD28 induced expression of mir-31 is increased by T-Bet and IFN-γ. (A) MiR-31 expression kinetics normalized to snU6 after the first activation of naive CD4 + (left panel) or reactivation of Th1 rep cells (right panel) with αCD3/28, presented relative to values obtained from naive CD4 + cells ex vivo , determined by qRT-PCR. Data is shown as mean ±SEM, n = 8–12 pooled from 3 to 4 independent experiments (One-way Anova with Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (B) RNA-Seq coverage of the miR-31 gene locus in Th1 rep cells (upper row) and analysis of published ChIP-seq data from Th1 cells for p300 ( 28 ), H3K4me3 and H3K27me3 ( 29 ) using the Cistrome Browser (lower row). (C) Analysis of the putative promoter region as determined in (B) using published ChIP-Seq data obtained from Th1 cells for T-Bet ( 34 ), STAT1 ( 28 ), and STAT4 ( 33 ) and from naive CD4 + T cells ( 35 ), as well as predicted conserved binding sites for these transcription factors obtained from ECR Browser. (D) Schematic overview of the murine miR-31 gene locus and the resulting primary transcript as analyzed in (B) . (E) MiR-31 expression normalized to Hprt in naive CD4 + cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ± IFN-γ (10 ng/ml), presented relative to values obtained from naive CD4 + cells ex vivo determined by qRT-PCR. Data is shown as mean ±SEM, n = 4–8 pooled from two independent experiments (One-way Anova with Dunn's test for multiple comparison, ** p ≤ 0.01).

    Journal: Frontiers in Immunology

    Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

    doi: 10.3389/fimmu.2018.02813

    Figure Lengend Snippet: TCR/CD28 induced expression of mir-31 is increased by T-Bet and IFN-γ. (A) MiR-31 expression kinetics normalized to snU6 after the first activation of naive CD4 + (left panel) or reactivation of Th1 rep cells (right panel) with αCD3/28, presented relative to values obtained from naive CD4 + cells ex vivo , determined by qRT-PCR. Data is shown as mean ±SEM, n = 8–12 pooled from 3 to 4 independent experiments (One-way Anova with Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (B) RNA-Seq coverage of the miR-31 gene locus in Th1 rep cells (upper row) and analysis of published ChIP-seq data from Th1 cells for p300 ( 28 ), H3K4me3 and H3K27me3 ( 29 ) using the Cistrome Browser (lower row). (C) Analysis of the putative promoter region as determined in (B) using published ChIP-Seq data obtained from Th1 cells for T-Bet ( 34 ), STAT1 ( 28 ), and STAT4 ( 33 ) and from naive CD4 + T cells ( 35 ), as well as predicted conserved binding sites for these transcription factors obtained from ECR Browser. (D) Schematic overview of the murine miR-31 gene locus and the resulting primary transcript as analyzed in (B) . (E) MiR-31 expression normalized to Hprt in naive CD4 + cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ± IFN-γ (10 ng/ml), presented relative to values obtained from naive CD4 + cells ex vivo determined by qRT-PCR. Data is shown as mean ±SEM, n = 4–8 pooled from two independent experiments (One-way Anova with Dunn's test for multiple comparison, ** p ≤ 0.01).

    Article Snippet: Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′.

    Techniques: Expressing, Activation Assay, Ex Vivo, Quantitative RT-PCR, MANN-WHITNEY, RNA Sequencing Assay, Chromatin Immunoprecipitation, Binding Assay

    MiR-31 is upregulated in murine Th1 rep cells, and in memory Th cells from the synovial fluid of RA patients. (A) MiR-31 expression in once (day 6) and repeatedly (three rounds of restimulation with 6 day intervals) activated Th1, Th2, Th17, and ex vivo isolated naive CD4 + cells normalized to snU6 determined by qRT-PCR. Each data point represents an independent experiment ( n = 12 [naive and Th1], 5 [Th2], 4 [Th17]) (Wilcoxon-Test for paired data, *** p ≤ 0.001). (B) MiR-31 expression normalized to snU6 in CD3 + CD4 + CD14 − CD45RO + T cells isolated from the synovial fluid of patients suffering from RA or blood from healthy control (HC) donors ex vivo or after 3 h of restimulation with PMA/ionomycin (P/I) ( n = 5 RA; n = 4 HC) determined by qRT-PCR. Each data point represents an individual donor, horizontal bar: median (Mann-Whitney test for unpaired data, * p ≤ 0.05).

    Journal: Frontiers in Immunology

    Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

    doi: 10.3389/fimmu.2018.02813

    Figure Lengend Snippet: MiR-31 is upregulated in murine Th1 rep cells, and in memory Th cells from the synovial fluid of RA patients. (A) MiR-31 expression in once (day 6) and repeatedly (three rounds of restimulation with 6 day intervals) activated Th1, Th2, Th17, and ex vivo isolated naive CD4 + cells normalized to snU6 determined by qRT-PCR. Each data point represents an independent experiment ( n = 12 [naive and Th1], 5 [Th2], 4 [Th17]) (Wilcoxon-Test for paired data, *** p ≤ 0.001). (B) MiR-31 expression normalized to snU6 in CD3 + CD4 + CD14 − CD45RO + T cells isolated from the synovial fluid of patients suffering from RA or blood from healthy control (HC) donors ex vivo or after 3 h of restimulation with PMA/ionomycin (P/I) ( n = 5 RA; n = 4 HC) determined by qRT-PCR. Each data point represents an individual donor, horizontal bar: median (Mann-Whitney test for unpaired data, * p ≤ 0.05).

    Article Snippet: Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′.

    Techniques: Expressing, Ex Vivo, Isolation, Quantitative RT-PCR, MANN-WHITNEY

    Immunohistochemistry using human anti-CD68 for macrophages for epicardial, mediastinal and subcutaneous adipose tissues of obese CAD and control groups. CD68+ cells (macrophages) are observed in the epicardial, mediastinal and subcutaneous adipose tissues of obese CAD group ( A , B and C ) and control group ( D , E and F ). CD68+ cells (upwards arrow) between normal and large adipocytes (black asterisks) are identified in the adipose tissues. These findings were endorsed by qRT-PCR of CD68 mRNA expression in all three adipose tissues of the study group. Magnification: x 40 for A , B , C , D , E and F.

    Journal: Cardiovascular Diabetology

    Article Title: The role of mediastinal adipose tissue 11?-hydroxysteroid d ehydrogenase type 1 and glucocorticoid expression in the development of coronary atherosclerosis in obese patients with ischemic heart disease

    doi: 10.1186/1475-2840-11-115

    Figure Lengend Snippet: Immunohistochemistry using human anti-CD68 for macrophages for epicardial, mediastinal and subcutaneous adipose tissues of obese CAD and control groups. CD68+ cells (macrophages) are observed in the epicardial, mediastinal and subcutaneous adipose tissues of obese CAD group ( A , B and C ) and control group ( D , E and F ). CD68+ cells (upwards arrow) between normal and large adipocytes (black asterisks) are identified in the adipose tissues. These findings were endorsed by qRT-PCR of CD68 mRNA expression in all three adipose tissues of the study group. Magnification: x 40 for A , B , C , D , E and F.

    Article Snippet: Gene expression analysis of 11β-HSD-1, GCR and CD68 mRNA expression levels of 11β-HSD-1, GCR and CD68 were determined by SYBR green-based qRT-PCR using a LightCycler (Roche-Germany) instrument.

    Techniques: Immunohistochemistry, Quantitative RT-PCR, Expressing

    Expression of NF-κB1 and Bcl-2 detection in glioma tissues by qRT-PCR. Notes: The expression of NF-κB1 in glioma was significantly higher than that in nonneoplastic brain tissues ( P

    Journal: OncoTargets and therapy

    Article Title: Nuclear factor-kappa B1 inhibits early apoptosis of glioma cells by promoting the expression of Bcl-2

    doi: 10.2147/OTT.S144014

    Figure Lengend Snippet: Expression of NF-κB1 and Bcl-2 detection in glioma tissues by qRT-PCR. Notes: The expression of NF-κB1 in glioma was significantly higher than that in nonneoplastic brain tissues ( P

    Article Snippet: Relative levels of NF-κB1 and Bcl-2 mRNA were examined using SYBR green quantitative real-time polymerase chain reaction (qRT-PCR) (LightCycler® 480; Hoffman-La Roche Ltd., Basel, Switzerland) and were normalized to levels of GAPDH mRNA.

    Techniques: Expressing, Quantitative RT-PCR