EMBO/SourceData huggingface dataset:
Figure 6CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (A) WT or EGR4-/- cells were loaded with Fura-2AM and plated on anti-CD3/CD28 antibodies for 2 hrs. Senicapoc (4 μM) or Tram-34 (5 μM) were added where marked by the arrow.CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (B) CD4+ T cells were isolated by negative selection from WT or EGR4-/- mice and incubated with anti-CD3/CD28 antibodies for 6 hrs in the presence or absence of either senicapoc, TRAM-34 or Shk-Dap22 before measuring expression of IFNγ levels by FACS analysis. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates. *** p < 0.001; **** p < 0.0001.CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (C) To measure the effect of KCa3.1 inhibition of cell proliferation, WT and EGR4-/- CD4+ T cells were isolated from the spleen by cell sorting and stained with CFSE. Cells were incubated with anti-TCRβ antibodies in the presence of vehicle, Senicapoc (5 µM) or TRAM-34 (5 µM) for 4 days and then collected for FACS analysis. (D) Generation analysis was completed for all data using FlowJo software. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates.
Figure 7B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (A) Luciferase expression was monitored by IVIS imaging at the indicated timepoints after luciferin injections. B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (B-D) Mice were sacrificed on day 20 and tumors were counted. Data is presented as total/mouse (B), N=9 for each gender and genotype. Differences in luciferase activity measured by IVIS in Panel A were determined by two-way ANOVA. Changes in luciferase activity were significantly altered in EGR4 knockout mice (P < 0.0112).B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. Mice were sacrificed on day 20 and tumors were counted. (B), lung tumors (C) and metastatic tumors (all tumors found outside of the lungs; D). N=9 for each gender and genotype. Differences in luciferase activity measured by IVIS in Panel A were determined by two-way ANOVA. Changes in luciferase activity were significantly altered in EGR4 knockout mice (P < 0.0112).B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45+ cells. WT lung tumors were 5.407 ± 1.843% CD45+, while EGR4KO mice were 12.445 ± 2.239% CD45+. WT spleens were 83.92 ± 3.344% CD45+, while EGR4KO mice were 95.13 ± 1.856% CD45+. Relative distributions of T and myeloid cells within the CD45+ populations were determined by flow cytometric analysis. (E,F) Distributions of T and myeloid cells found within lung tumors (E) vs. the spleen (F).B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45+ cells. (G) Representative FACS plots depicting gating strategies for defining Treg cell populations in Panels E and F
Figure 8Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (A) CD4+ and CD8+ T cells were stimulated in vitro with ionomycin/PMA before intracellular staining for IFNγ, IL-17 and IL-9 and FACS analysis.Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (B,C) Representative FACS plots depicting gating strategies for defining cytokine expression in panels A to DTumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (D) Percentages of each population.Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (E) Gating strategies.Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (F) FR4 and PD1 levels in WT vs. EGR4-/- T cells.
Figure 1a-c, Expression of CD45RO and CCR7 (a), CD45RO and CD27 (b), CD27 and CD28 (c) on peripheral blood CD8 T cells of three participants [P1 (heterozygous mutation), P2 (homozygous mutation) and P3 (heterozygous) for the c.192G>C mutation in the DJ-1 gene]. d-g, Expression of CD57 (d), PD-1 (e), EOMES (f) and T-bet (g) on peripheral blood CD8 T cells of three participants. The enlarged number in the corresponding gate represents the corresponding percentage out of the parent population. h
Figure 2b-c, Percentages of CD44low CD62Lhigh (Tn) (b) and CD44high CD62Llow (Tem) (c) cells among total CD8 T cells of spleen and pLNs from young and 45-wk-old Dj-1 KO and WT littermates (young KO, n=5; young WT, n=5; 45-wk-old KO, n=8; 45-wk-old WT, n=6; for 45-wk-old mice, data pooled from 2 independent experiments).e, Percentages of Ki-67+ cells among total CD8 T cells. f, Percentage of Ki-67+ cells among splenic CD8 Tn, Tem and Tcm in 45-wk-old Dj-1 KO and age- and sex-matched WT mice. gj, Percentages of Tn (top), Tvm (middle), Tmem (bottom) among 60-wk-old or young mice (young KO, n=4; young WT, n=3; 60-wk-old KO, n=4; 60-wk-old WT, n=5).
Figure 3b, Percentages of KLRG1+ CD8 T cells derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO (n=4) or WT recipients (n=5). c, Percentages of KLRG1+ among CD8 Tem derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO or WT recipients. d d, Percentages of PD-1+ population among CD8 T cells derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO (n=4) or WT recipients (n=5).j, Percentage of KLRG1+ population among total CD8 T cells (55-wk-old KO, n=4; 55-wk-old WT, n=5; young KO, n=4; young WT, n=4). k, Percentage of KLRG1+ population among CD8 Tem cells. l, Percentage of KLRG1+PD-1+ population among total CD8 T cells (55-wk-old KO, n=4; 55-wk-old WT, n=5; young KO, n=4; young WT, n=4). m, Percentage of KLRG1+PD-1+ population among CD8 Tem cells. Dat
Figure 4b, the percentages of proliferating cells among living CD8 singlets (CD8 Tn were from young mice).e, the percentages of proliferating cells among living CD8 singlets (CD8 Tn were from 45-wk-old mice).
Figure 5e-h, Oxygen consumption rate (OCR) of unstimulated (e) and stimulated CD8 Tn (f) by 2ug/ml of anti-CD3 and 1ug/ml of anti-CD28 for 24 hours. The CD8 Tn were isolated from 45-wk-old mice. OCR of unstimulated (g) and stimulated CD8 Tn (h) by anti-CD3 and -CD28 for 24 hours. The CD8 Tn were isolated from young mice. Left of each OCR measurement panel, representation plots following Mito stress test. Right of each panel, quantitation of basal and maximum respiration under Mito stress test conditions The dashed line indicates the time when the corresponding modulator Oligomycin (Oligo), Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) or Rotenone and Antimycin (AA/Rot) was added.i-k, Extracellular acidification rate (ECAR) of unstimulated and stimulated CD8 Tn by anti-CD3 and -CD28 antibodies for 24 hours (i). The CD8 Tn were isolated from 45-wk-old mice. ECAR of unstimulated (j) and stimulated (k) CD8 Tn by anti-CD3 and -CD28 for 24 hours. The CD8 Tn were isolated from young mice. Left of each ECAR measurement panel, representation plots following Glycolysis stress test. Right of each panel, quantitation of glycolysis and glycolytic capacity under Glycolysis stress test conditions. The dashed line indicates the time when the corresponding modulator glucose, Oligomycin (Oligo) or 2-deoxy-glucose (2-DG) was added.
Figure 1(C) Levels of mucosal vtRNA2-1, vtRNA1-1, vtRNA1-2, and vtRNA1-3 in the colon of patients with active ulcerative colitis (UC). Values are the means ± SEM (n = 6 per group). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with controls. (D) Levels of tissue vtRNAs in the ileal mucosa of patients with Crohn's disease (CD). Values are the means ± SEM (n = 6 per group). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with controls.
Figure 2(B) Expression levels of claudin 1, occludin, E-cadherin, ZO-1, JAM-A, and MVP in cells described in A. Hsc70 immunoblotting was performed as an internal control for equal loading.
Figure 3(B) Immunoblots of claudin 1, occludin, E-cadherin, ZO-1, and JAM-A in cells treated as described in A. Hsc70 immunoblotting served as an internal control for equal loading. Three separate experiments were performed and showed similar results.
Figure 4(E) Immunostaining (left) of Paneth (lysozyme-positive) cells and quantitative data (right) in intestinal organoids treated as described in A. Lysozyme: green. Scale bars, 100 µm. Values are means ± SEM (n = 4 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control vector.
Figure 5(B) Immunoblots of claudin 1, occludin, JAM-A, ZO-1, and E-cadherin in the mucosa from mice treated as described in A. GAPDH immunoblotting served as an internal control for equal loading.
Figure 6(C) Newly synthesized claudin 1 and occludin proteins in cells treated as described in A. After cells were exposed to L-azidohomoalaine (AHA), cell lysates were incubated with the reaction buffer containing biotin/alkyne reagent; the biotin-alkyne-azide-modified protein complex was pulled down by paramagnetic streptavidin-conjugated dynabeads. (D) Quantitative data of immunoblots shown in C. Values are means ± SEM (n = 3 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control vector.
Figure 7(A) Association of vtRNA2-1 with HuR in Caco-2 cells as measured by biotin-labeled RNA pull-down assays. After cytoplasmic lysates were incubated with biotinylated vtRNA2-1, levels of HuR and other proteins in the pull-down material were assessed. Three separate experiments showed similar results.(D) Changes in the expression levels of claudin-1, occludin, and E-cadherin proteins 48 h after cells were transfected with HuR expression vector alone or co-transfected with HuR and vtRNA2-1 expression vectors.
Figure 1A. Relative abundance of miR species in human naive CD4 T cells. Cell purity was >97%.B, C. Average miR expression levels in unstimulated naive CD4 T cells non-infected vs HIV-1NL4-3 (B) or HIV-2ROD (C) infected after 24h (n=2, three pooled samples per library). Mean normalized read counts across libraries are shown. Library normalization was performed using the mean normalization method available in the Deseq package of Bioconductor (Anders Huber, 2010). Lines indicate changes of +/- 1 log2 fold between samples.
Figure 2A. Average miR expression levels in naive CD4 T cells (purity >97%) before and after 72h TCR-stimulation. Lines indicate changes of +/- 1 log2 fold between samples (n=2, paired pools of three and nine individual samples per library). Library normalization performed using the mean normalization method available in Deseq package of Bioconductor (Anders Huber, 2010). miRs with significant changes are highlighted (black) and with insets presenting mean read counts in unstimulated samples (mean), log2 fold change values in stimulated samples (log FC) and corresponding adjusted p value (p; Fisher test implemented in Deseq). The two most highly expressed miRs (mir-21-5p and miR-146b-5p) are also indicated.B. Relative miR-34c-5p and miR-155-5p expression levels (2deltaCt normalized to RNU48) in unstimulated and stimulated naive CD4 T cells of individual samples pooled to generate the sequencing libraries. mean ± SEM and p-value for differences in miR-155-5p expression (paired two tailed t-test) are shown. Comparison could not be performed for miR-34c-5p as it is undetermined in unstimulated samples.C. Time course quantification of miR-34c-5p expression (top panel, 2deltaCt values) in three different donors.C. Time course quantification of cell proliferation (middle panel) and cell activation (bottom panel) markers by flow cytometry, in three different donors.
Figure 3A, B. Comparison of the mean miR expression level by in TCR-stimulated naive CD4 T cell small RNA-seq libraries from uninfected and HIV-1NL4-3 (A) or HIV-2ROD (B) infected samples (24h). Only miRNAs with a minimum of 10 normalized read counts. Library normalization was performed using the mean normalization method available in Deseq package of Bioconductor (Anders Huber, 2010). Lines indicate changes of +/- 1 log2 fold between samples (n=3, paired pools of three individual samples per library). miR-34c-5p is highlighted in black.C. Quantification of miR-34c-5p expression levels of the individual samples pooled to generate the sequencing libraries by qPCR (mean±SEM 2deltaCt values and p value from a paired two tailed t test; n=5).
Figure 4A. Cell-associated viral DNA levels (left), Tat mRNA levels (center), and RT activity levels (right) in cell culture supernatants in infected J34c cells vs. infected JØ cells at 24 and 48 hours post-infection. * p0.05 (paired two tailed t test).B. Representative flow cytometry plot of intracellular HIV-1 Gag protein staining (KC57) versus side scatter (SSC) in JØ and J34c cells, mock infected or infected with HIV-1NL4-3, after 48 hours of culture and corresponding fold change (J34c/JØ) plot in four biological replicate assays. Differences to control were found to be significant with a paired two tailed t test (p0.05).C. Fold change levels for miR-34c-5p, cell-associated viral DNA level, Tat mRNA, and RT activity levels in J34c cells treated with miR-34c-5p antagomiR versus control antagomiR, 48 hours post-infection. Differences to control were found to be significant with a paired two tailed t test (p0.05).
Figure 5A. Hierarchical clustering of the 82 differentially expressed (DE) genes identified in Jurkat cell lines overexpressing miR-34c-5p (J34c-2 and J34c-5) vs control (JØ-1, 2 and 3 clones). Color scale represents mean centered log2 fold expression level across samples. Pie charts represent the relative proportion of predicted miR-34c-5p binding sites by the Miranda or PITA algorithms (or both) in down-regulated (top) and up-regulated (bottom) gene sets.B. qRT-PCR validation of selected genes. Plot represents the average percentage of target gene mRNA (normalized to GAPDH) in J34c vs JØ cells (all differences were found to be significant with a paired two tailed t test, P<0.05; n=5).
Figure 7B. Western-blot of PCAF/KAT2B and histone H3 in JØ or J34c (top), confirming decrease in protein levels.B. Bottom: antagomiR to miR-34c-5p results in increased KAT2B mRNA levels compared to control-treated cells. Plot presents relative expression values for mRNA in 3 biological replicates; bars represent mean and SEM. Differences were found to be significant with p<0.05 (paired two tailed t test).C. Predicted target sites for miR-34c-5p in the PCAF/KAT2B mRNA(black bars) and miR-target hybridization for the two strongest sites (top); Luciferase assay with the pEZX renilla/firefly-PCAF/KAT2B 3'UTR reporter in HEK293 cells co-transfected with either the miR-34c-5p overexpression vector or pcDNA3 empty control (bottom). Differences were found to be significant with p<0.05 (paired two tailed t test, n=5).
Figure 1Components of the RLCco-precipitate with ER markers in density gradient fractionation. The post-nuclear supernatant of cortical neurons was loaded underneath a continuous Optiprep density gradient. ER markers Calnexin, Ribophorin I and Climp63, co-sedimented with ribosomes (RPS6, ribosomal protein subunit 6), mitochondria (Tim23) and RLC proteins at fractions 3-5. The bottom graph shows the density of each fraction.B) TRBP, PACT and Ago2 partially co-localize with the ER marker mCherry-CLIMP63. Hippocampal neurons (10 DIV) expressing mCherry-CLIMP63 were immunostained with TRBP, PACT or Ago2 antibodies. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 10μm).C) GFP-Dicer partially co-localizes with mCherry-CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons. Neurons were imaged at 10 DIV using total internal reflection fluorescence (TIRF) microscopy using a penetration depth of 150nm. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 2μm or 10μm).D) The majority of Dicer, TRBP and PACT are associated with neuronal membranes. Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons. The relative quantity of depicted proteins was analyzed in three independent experiments (p<0.005, t-test type 3, n=3, error bars s.d.). Actin and ribophorin I were used as loading controls for cytoplasmic and membrane fractions, respectively.E) TRBP is cytoplasmic in primary astrocytes. Sequential detergent extraction was performed in primary astrocyte cultures. Actin and calnexin were used as loading controls for cytoplasmic and membrane fractions, respectively.F) The majority of Dicer, TRBP and PACT proteins are present in cytoplasmic fractions in HEK293T cells, isolated using sequential detergent extraction.
Figure 2A) BDNF stimulation leads to a decrease in TRBP levels from ER microsomes. Cortical neurons were stimulated BDNF with or vehicle control and ER microsomes were isolated using differential centrifugation. Protein intensity values were normalized to calnexin in each of three independent experiments (p=0.008, t-test type 3, n=3, error bars; s.d.).B) Endogenous TRBP partially co-localizes with calnexin at the neuronal cell soma and primary dendrites. Hippocampal neurons were transfected with GFP at 7DIV and fixed at 10DIV following BDNF or control stimulation. Boxed insets are magnifications of the primary dendrite (15μm in length). Arrows point to overlapping puncta (scale bars; 20μm (GFP) or 10μm).C) BDNF leads to a decrease in co-localization between TRBP and calnexin at the soma and primary dendrites of hippocampal neurons. Manders co-localization co-efficients between TRBP and Calnexin were measured in the cell soma or primary dendrites (15μm) using GFP as a morphological mask. Average co-localization values were normalized to controls in each experiment (p<0.008, t-test type 3, n=4, error bars; s.d.).D) BDNF decreases mCherry-CLIMP63 co-localization with TRBP, but not with PACT or Ago2. Hippocampal neurons were transfected with GFP and mCherry-CLIMP63 at 7DIV and immunostained three days later, following BDNF or control stimulation. Manders co-localization was measured at the cell soma using GFP as a mask as indicated (p=0.01, t-test type 3, n=4, error bars; s.d.).E) BDNF leads to an increase in the levels of TRBP (p=0.03) and PACT (p=0.004) in the cytoplasmic fraction. Cortical neurons (7 DIV) were control- or BDNF-stimulated and cytoplasmic fractions were isolated using digitonin. Protein intensity values were normalized to actin in each experiment. The position of the star corresponds to the TRBP band used for analysis (~43kDa) (t-test type 3, n=4, error bars; s.d.).F) The BDNF-induced increase in cytoplasmic TRBP is dependent on intracellular calcium. Cortical neurons (7 DIV) were treated with vehicle control, BDNF and BAPTA-AM as indicated, and cytoplasmic fractions were isolated using digitonin. Protein intensity values were normalized to actin in each independent experiment (p=0.03, n=3, error bars; s.d.).
Figure 3BDNF stimulation causes a decrease in the TRBP-Dicer interaction. Young cortical neurons were treated with BDNF or vehicle control and co-immunoprecipitations (co-IPs) were performed using 2μg control IgG or polyclonal anti-TRBP antibodies. Dicer binding in co-IP was normalized to inputs in each experiment (p=0.001, t-test type 2, n=3, error bars represent s.d.).B) Short BDNF stimulation changes the levels of only a few microRNAs, such as miR-16-5p and miR-551b-5p. Volcano plot of small RNA sequencing data from BDNF- or control-treated cortical neurons (6DIV) was based on differential expression analysis using the edgeR package. Individual points represent the average fold change obtained in three independent experiments for each miRNA, and plotted against obtained p-values. MiRNAs above the red dashed line are significant (p<0.05, t-test type 2, n=3).C) BDNF stimulation leads to a transient decrease in mature miR-16-5p levels. 6DIV cortical neurons were treated with BDNF for 10 or 20 minutes and mature miRNA levels were measured in rtPCR. Ct values for each miRNA were normalized to U6 and to 20 minutes of control stimulation (n=3, p=0.02, t-test type 3, error bars; s.d.).D) BDNF stimulation leads to a transient increase in the levels of pre-miR16 after 10 minutes of BDNF stimulation. Ct values obtained in rtPCR were normalized to U6 and to 20 minutes control stimulation (n=4, t-test type 3, error bars; s.d.).E) BDNF causes a decrease in pre-miR16 binding to TRBP. Cortical neurons were treated with BDNF or vehicle control and RNA IPs were performed using a mouse monoclonal anti-TRBP antibody. Ct values measured in rtPCR were normalized to respective inputs (n=3, p=0.002, t-test type 3, error bars represent s.d.).
Figure 4A) BDNF changes the canonical isomiR proportion of a subset of miRNAs. IsomiR levels were compared to the total amount of the corresponding miRNA. Shown are the miRNAs that had significant changes in the canonical isomiR (annotated as '0', p<0.05, t-test type 2, n=3). Results are shown in order of decreasing significance. Shown in red are miRNAs of the miR16 family, miR-16-5p and miR-15b-5p. Black squares correspond non-existing isomiRs. Numbers on the top of the table represent the trimming (negative) or addition (positive) of nucleotides to the 3'- or 5'-end of the canonical 5p or 3p isomiRs, respectively. Only templated nucleotide additions and trimmings, which might reflect Dicer-mediated pre-miRNA cleavage, were considered for analysis.B) BDNF stimulation leads to a decrease in the canonical (22 nucleotides) isomiRs of the miR16 family, miR-16-5p (p=0.005) and miR-15b-5p (p=0.02). In both cases, this is accompanied by an increase in the levels of shorter isomiRs; the 19-nucleotide isoform of miR-16-5p (p=0.04) and the 21-nucleotide isoform of miR-15b-5p (p=0.007). The relative isomiRs proportion was calculated as described in A (t-test type 2, n=3, error bars; s.d.).