Journal: Nature Communications

Article Title: Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis

doi: 10.1038/ncomms7253

Figure Lengend Snippet: ( a ) Flow cytometric stains from Roquin san/san and Roquin +/+ T cells showing the gating strategy used to sort CD4 + CD44 lo CD25 − naive T cells. ( b ) Scatter plot of microRNA expression in sorted naive T cells obtained by microarray analysis. ( c ) qRT–PCR showing relative expression levels of mature miR-146a and miR-21 in naive T cells, normalized to U6. ( d ) Relative expression levels of miR-146a measured by qRT–PCR in Rc3h1 fl/fl ;Rc3h2 fl/fl ; CD4-Cre double knockout naive T cells normalized to snoRNA202. ( e , f ) qRT–PCR analysis of miR-146a ( e ) and miR-21 ( f ) in Roquin san/san .Ly5b: Roquin +/+ .Ly5a or of Roquin +/+ .Ly5b: Roquin +/+ .Ly5a mixed chimeras. Each dot represents an individual mouse and the bar represents the median value in each group. U -test: * P <0.02, ** P <0.05, *** P <0.005. See also .

Article Snippet: Rc3h1 fl/fl ;Rc3h2 fl/fl ; CD4-Cre mice were housed in a pathogen-free barrier facility at the Helmholtz Zentrum, München.

Techniques: Expressing, Microarray, Quantitative RT-PCR, Double Knockout

Journal: Nature Communications

Article Title: Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis

doi: 10.1038/ncomms7253

Figure Lengend Snippet: ( a ) Relative expression of pri-miR-146a in naive T cells sorted from Roquin +/+ and Roquin san/san mice measured by qRT–PCR using three different sets of primers shown as (i), (ii) and (iii) in the schematic. NS, not significant ( P <0.05, U -test). ( b ) The levels of pre-miR-146a in Roquin +/+ and Roquin san/san naive T cells measured by qRT–PCR. ( c ) Northern blots showing precursor and mature miRNA transcripts (arrows) of miR-146a and miR-150 in naive T cells of Roquin san/san and Roquin +/+ mice. U6 snRNA was used as a loading control and is shown below the miR-146a northern blot. Weaker less abundant bands are miRNA decay products. M, marker. ( d ) Relative expression of miR-146a in Dicer-floxed-Rosa26-Cre-ER MEFs ( Dicer1 −/− ) and CD4-CRE (wt) control MEFs assessed by qRT–PCR. MEFs were retrovirally transduced with Roquin wt and Roquin san containing a GFP reporter or with an empty GFP vector control. After 2 days, MEFs were treated with tamoxifen to delete Dicer. GFP-positive cells were sorted 7 days later. Each dot represents individual mice ( a , b ) or technical replicates ( d ) and the bars represent the median value in each group. U -test: * P <0.05, ** P <0.005. Results are representative of two independent experiments.

Article Snippet: Rc3h1 fl/fl ;Rc3h2 fl/fl ; CD4-Cre mice were housed in a pathogen-free barrier facility at the Helmholtz Zentrum, München.

Techniques: Expressing, Quantitative RT-PCR, Northern Blot, Control, Marker, Transduction, Plasmid Preparation

Journal: Nature Communications

Article Title: Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis

doi: 10.1038/ncomms7253

Figure Lengend Snippet: ( a ) In vitro processing of pre-miR-146a in HEK293T cells after transfection with FLAG-Dicer (lane 1), Roquin wt -GFP (lane 3) or Roquin san -GFP (lane 4). Co-transfection of FLAG-Dicer with either Roquin wt -GFP (lane 5) or Roquin san -GFP (lane 6) is included. No transfection control is shown in lane 2. ( b ) In vitro processing of pre-miR-146a in HEK293T cells after transfection with FLAG-Dicer (lane 2) alone, or co-transfection of FLAG-Dicer with either Roquin wt -GFP (lane 3) or Roquin san -GFP (lane 4). Lane 1: No transfection control. The lower panels show densitometric analysis of the northern blots to quantify 32 P. The ratios of the mature versus precursor miRNA intensities are plotted. Intermediates or low abundance compared with the fully digested products were not included in the quantification.

Article Snippet: Rc3h1 fl/fl ;Rc3h2 fl/fl ; CD4-Cre mice were housed in a pathogen-free barrier facility at the Helmholtz Zentrum, München.

Techniques: In Vitro, Transfection, Cotransfection, Control, Northern Blot

Journal: Nature Communications

Article Title: Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis

doi: 10.1038/ncomms7253

Figure Lengend Snippet: ( a ) miR-146a in Roquin san/san and Roquin +/ + naive T cells measured by qRT–PCR and normalized to comparatively stable U6 snRNA. The cells were treated with actinomycin D (10 mg ml −1 ) for the times indicated. The amount of miR-146a at 0 h was assigned 100%. The error bar represents the range of the data for two experiments. ( b ) Pre-miR-146a was transfected along with Roquin wt -GFP or Roquin san -GFP in HEK293T cells and qRT–PCR for miR-146a was performed on GFP-positive cells on days 2 and 5 post transfection. See also . ( c ) miR-146a in MEFs 7 days after retroviral transduction of precursor miR-146a along with Roquin wt or Roquin san . Each dot represents a technical replicate and the bars represent the median value in each group. U -test: * P <0.01, ** P <0.001, NS, not significant. The data are representative of two independent experiments.

Article Snippet: Rc3h1 fl/fl ;Rc3h2 fl/fl ; CD4-Cre mice were housed in a pathogen-free barrier facility at the Helmholtz Zentrum, München.

Techniques: Quantitative RT-PCR, Transfection, Retroviral, Transduction

Journal: Nature Communications

Article Title: Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis

doi: 10.1038/ncomms7253

Figure Lengend Snippet: ( a ) Normalized counts per million (CPM) of miR-146a and miR-150 in sRNA deep-sequencing from Roquin san/san versus Roquin +/+ T cells. ( b ) The ratio of non-templated addition of nucleotides at +1 position in Roquin san/san and Roquin +/+ T cells. A, adenine; C, cytosine; G, guanine and U, uracil. The two ‘hinges’ are the first and third quartile, the notches extend to ±1.58 × interquartile range/sqrt(no. of observations) and whiskers extend to the data range. ( c ) The percentage of mono-uridylated to exactly matching mature miR-146a or miR-150 was calculated from Roquin +/+ and Roquin san/san littermates using the sRNA deep sequencing data set, the percentage change was calculated. sRNA: small RNA. ( d ) qRT–PCR analysis of endogenous miR-146a and U6 in immunoprecipitates from total human tonsil lymphocytes using anti-IgG, no antibody, anti-Roquin IgG (Roquin) and anti-Roquin IgG pre-incubated with a blocking Roquin peptide (Roquin-pep). 10% of the whole cell lysate (total input) was removed before immunoprecipitation and the results are presented as percentage of total input. ** P =0.05 ( U -test), NS, not significant. Each dot represents a technical replicate. The results are representative of five independent experiments. ( e , f ) SPR study of the binding of RoquinM199R 1–484 and RoquinWT 1–484 to immobilized 5’-biotinylated pre-miR-146a. ( e ) Blank-subtracted Biacore sensograms for RoquinM199R 1–484 (twofold dilutions, 1,000–3.9 nM) and RoquinWT 1–484 (2,000–7.8 nM) binding to pre-miR-146a. Protein concentrations increased from the bottom to the top curve. ( f ) Binding isotherms for equilibrium responses. The solid curves were calculated from the derived K D values for a 1:1 interaction of 110±20 (for M199R) and 370±50 nM (WT). Fit R max values were 330±20 (for WT) and 242±13 response units (M199R).

Article Snippet: Rc3h1 fl/fl ;Rc3h2 fl/fl ; CD4-Cre mice were housed in a pathogen-free barrier facility at the Helmholtz Zentrum, München.

Techniques: Sequencing, Quantitative RT-PCR, Incubation, Blocking Assay, Immunoprecipitation, Binding Assay, Derivative Assay

Journal: Nature Communications

Article Title: Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis

doi: 10.1038/ncomms7253

Figure Lengend Snippet: ( a ) Structural and schematic representations of Roquin 1–484 . (i) Overall architecture of Roquin 1–484 (RING domain=red, ROQ domain=beige, HEPN=light blue, additional helix packing against the HEPN domain=dark blue. (ii) Top: Schematic of the known domain boundaries of Roquin before this work, middle/bottom: domain boundaries revealed by the crystal structure. Note that the ROQ domain is an insertion in the HEPN domain. The ZnF (CCCH) domain was present in the crystallized protein but is not visible in the crystal structure. ( b ) Electrostatic surface diagram of Roquin 1–484 (180 degrees rotated left to right), highlighting positively charged surfaces likely to be involved in RNA binding. The surface indicated in the right hand panel is in a very similar location to the surface used by ADAR1 to bind nucleic acids. The orientation in the left hand panel is identical to that in a (i). ( c ) Structural alignment of Roquin HEPN domain with HEPN domain from human Sacsin, (PDB ID:3O10, grey). ( d ) Structural alignment of Roquin ROQ domain (beige) with NusB (PDB ID: 2JR0, orange) and the winged helix-turn-helix from ADAR1 (PDB ID: 1QBJ, orange). ( e ) Structure of the Roquin RING domain and schematic showing the residues involved in coordinating the two zinc atoms. ( f ) Residue F234 is flipped out of the structure in the ROQ M199R mutant, becoming solvent exposed. left: wild-type (green); middle: M199R (beige); right: overlay. See also .

Article Snippet: Rc3h1 fl/fl ;Rc3h2 fl/fl ; CD4-Cre mice were housed in a pathogen-free barrier facility at the Helmholtz Zentrum, München.

Techniques: RNA Binding Assay, Residue, Mutagenesis, Solvent

Journal: Nature Communications

Article Title: Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis

doi: 10.1038/ncomms7253

Figure Lengend Snippet: ( a ) Immunoprecipitation of HEK293T cells transiently co-transfected with N-terminal V5-tagged Roquin and C-terminal FLAG-tagged Ago2; using anti-V5 and anti-FLAG antibodies. Half of the immunoprecipitate was treated with RNaseA. Roquin and Ago2 proteins were detected by western blot analysis using anti-V5 and anti-FLAG antibodies. Total lysate is shown in lane 1 as input. Mouse anti-CD71 antibody was used as a control. Anti-PABP antibody was used to confirm the RNase activity. The data are representative of three independent experiments. See also . ( b ) Quantification of proximity ligation assays showing average number of PLA spots per average number of cells (PLA/nuclei) for each field of view (represented by the individual dots in each column). At least five fields of view containing 50–100 cells or nuclei were imaged per experiment. Data are representative of four independent assays. U -test: ** P <0.002. ( c ) Confocal images of HEK293T cells showing molecular proximity of endogenous Roquin and Ago2 (left panel), Roquin and RCK (middle panel) and Roquin and GFP (right panel). Nuclei were stained with DAPI. Scale bar, 25 μm. See also . ( d ) Schematic of Roquin binding to miR-146a, Ago2 and Icos 3′-UTR containing CDE and miR-146a binding sites TS1 and TS2. See also . ( e ) Relative luciferase levels in mouse primary T cells retrovirally transduced with either intact full-length mouse Icos 3′-UTR, or this 3′-UTR mRNA carrying mutations in both miR-146a target sites, in the CDE or in both miR-146a target sites plus CDE. Each dot represents a technical replicate. U -test: *** P <0.001; ** P <0.01. This is a representative of three independent assays.

Article Snippet: Rc3h1 fl/fl ;Rc3h2 fl/fl ; CD4-Cre mice were housed in a pathogen-free barrier facility at the Helmholtz Zentrum, München.

Techniques: Immunoprecipitation, Transfection, Western Blot, Control, Activity Assay, Ligation, Staining, Binding Assay, Luciferase, Transduction

Journal: Cell Reports Medicine

Article Title: Development of a PCSK9-targeted nanoparticle vaccine to effectively decrease the hypercholesterolemia

doi: 10.1016/j.xcrm.2024.101614

Figure Lengend Snippet:

Article Snippet: Specific-pathogen-free (SPF) wild-type C57BL/6 mice (6–8 weeks old, female or male) and BALB/c mice (6–8 weeks old, female) were purchased from GemPharmatech Co., Ltd. Transgenic C57BL/6-hPCSK9 mice, Bcl6 fl/fl mice with loxP sites flanking the entire Bcl6 locus gene, CD4 cre transgenic mice, and LDLR knockout mice (LDLR −/− ) were also obtained from, and bred at, GemPharmatech Co., Ltd. CD4 cre mice were crossed with Bcl6 fl/fl mice to produce CD4 cre Bcl6 fl/fl conditional knockout (CKO) mice, which used the Bcl6fl/fl mice as controls.

Techniques: Recombinant, Virus, Enzyme-linked Immunosorbent Assay, Staining, Cell Stimulation, Adjuvant, Western Blot, Reverse Transcription, Plasmid Preparation, Protein Extraction, RNA Extraction, Software

Journal: Molecular Therapy

Article Title: Tim-3 regulates sepsis-induced immunosuppression by inhibiting the NF-κB signaling pathway in CD4 T cells

doi: 10.1016/j.ymthe.2021.12.013

Figure Lengend Snippet: The proportion of Tim-3 + CD4 T cells is increased in CD4 T cells from septic immunosuppression patients (A) Volcano plot of CD4 T cells from healthy volunteers and septic immunosuppression patients. The top five upregulated genes were Tim-3, XIST, TMEM38B, LIMS2, and IL10RB. The top five downregulated genes were ADA2, LDLRAD4, PRMT6, MMACHC, and TENM1. (B) Enriched gene ontology (GO) functions of upregulated genes in CD4 T cells. Upregulated genes in CD4 T cells of septic immunosuppression patients were enriched in immune-related pathways, especially lymphocyte- and leukocyte-related pathways, as well as several metabolic pathways. (C) Verification of the transcription levels of the top five upregulated and top five downregulated genes in CD4 T cells between sepsis patients and healthy volunteers (means ± SD; n = 30; ∗p < 0.05 and ∗∗p < 0.01 versus healthy control group, one-way ANOVA). (D) Tim-3 expression on CD4 T cells in 30 sepsis patients and 30 healthy volunteers by flow cytometry. The percentage of Tim-3-positive CD4 T cells (means ± SD; n = 30; ∗∗p < 0.01 versus healthy control group, one-way ANOVA). Tim-3 expression on CD4 T cells is presented as the mean fluorescence intensity (means ± SD; n = 30; ∗∗p < 0.01 versus healthy control group, one-way ANOVA).

Article Snippet: C57BL/6, Cd4 cre , Tim-3 fL/fL , and Tim-3 −/− mice were purchased from the Shanghai Model Organisms Center.

Techniques: Control, Expressing, Flow Cytometry, Fluorescence

Journal: Molecular Therapy

Article Title: Tim-3 regulates sepsis-induced immunosuppression by inhibiting the NF-κB signaling pathway in CD4 T cells

doi: 10.1016/j.ymthe.2021.12.013

Figure Lengend Snippet: Tim-3 knockout reduces sepsis mortality by preserving organ function (A) Survival curves of WT, Cd4 Cre Tim-3 fL/fL , and Tim-3 −/− mice in the CLP first hit and second hit models of sepsis. p = 0.0062 compared with WT and Cd4 Cre Tim-3 fL/fL and p = 0.008 compared with WT and Tim-3 −/− mouse in CLP second hit mouse of sepsis (n = 30 mice per group; Kaplan–Meier survival analysis). (B) Serum enzyme activity in the heart, liver, and kidney of CLP first and second hit mouse post 1, 3, and 7 days (n = 5; ∗p < 0.05 and ∗∗p < 0.01 versus WT, one-way ANOVA). (C) Secreted cytokines in WT, Cd4 Cre Tim-3 fL/fL , and Tim-3 −/− mouse peripheral blood were profiled using a Proteome Profiler™ Array Mouse XL Cytokine Array Kit. (D) Hematoxylin and eosin staining of WT, Cd4 Cre Tim-3 fL/fL , and Tim-3 −/− mouse lungs: alveolar septum thickening (blue arrow), leukocyte infiltration (blue arrowhead), alveolar congestion and edema (black arrow). Magnification, 200×; Scale bars, 100 μm.

Article Snippet: C57BL/6, Cd4 cre , Tim-3 fL/fL , and Tim-3 −/− mice were purchased from the Shanghai Model Organisms Center.

Techniques: Knock-Out, Preserving, Activity Assay, Staining

Journal: Molecular Therapy

Article Title: Tim-3 regulates sepsis-induced immunosuppression by inhibiting the NF-κB signaling pathway in CD4 T cells

doi: 10.1016/j.ymthe.2021.12.013

Figure Lengend Snippet: Tim-3 is correlated with a more severe exhaustion state of CD4 T cells during septic immunosuppression (A) The proliferation ability of Tim-3 + CD4 T cells and Tim-3 – CD4 T cells after CLP first hit (left) and second hit (right) is shown as the percentage of Ki67 + cells. Data are representative of three independent experiments with 10 mice per group in each experiment (p = 0.0006 in CLP second hit mouse of sepsis, one-way ANOVA). (B) Expression of LAG3 and 2B4 on Tim-3 + CD4 T cells and Tim-3 – CD4 T cells after CLP first hit and second hit models (n = 10; p < 0.01 in CLP second hit mouse of sepsis, one-way ANOVA). (C) Release of IL-10, TNF-α, IL-1β, and IL-6 in Tim-3 + CD4 T cells and Tim-3 − CD4 T cells in response to 100 ng/mL LPS stimulation in vitro for 24 h (p = 0.0008 for TNF-α, p = 0.0143 for IL-1β, p < 0.01 for IL-10 and IL-6, one-way ANOVA).

Article Snippet: C57BL/6, Cd4 cre , Tim-3 fL/fL , and Tim-3 −/− mice were purchased from the Shanghai Model Organisms Center.

Techniques: Expressing, In Vitro

Journal: Molecular Therapy

Article Title: Tim-3 regulates sepsis-induced immunosuppression by inhibiting the NF-κB signaling pathway in CD4 T cells

doi: 10.1016/j.ymthe.2021.12.013

Figure Lengend Snippet: HMGB1 is a ligand for Tim-3 on CD4 T cells in septic immunity (A) RT-PCR quantification of Gal-9 mRNA in the thymus, spleen, lung, liver, and kidney of CLP first hit and second hit model mice. The results are presented relative to the expression of GAPDH (n = 3; means ± SD; one-way ANOVA). (B) RT-PCR analysis of IL-1β, TNF-α, and IL-6 mRNA without or with rGal-9 or α-Gal-9 treatment for 12 h. The results are presented relative to GAPDH expression (n = 3; means ± SD; one-way ANOVA). (C) Administration of rGal-9 or α-Gal-9 to mice prevented CLP second hit-induced death (n = 20 mice per group; Kaplan–Meier survival analysis). (D) Binding of biotin-labeled rHMGB1 to plates coated with PBS or fusions of Fc-RAGE (RAGE-Fc) or Fc-Tim-3 measured by colorimetric analysis and presented as absorbance at 450 nm. (E) RT-PCR quantification of HMGB1 mRNA in the thymus, spleen, lung, liver, and kidney of CLP first hit and second hit mouse models. The results are presented relative to the expression of GAPDH (n = 3; means ± SD; p < 0.05 thymus versus lung, liver, or kidney in CLP second hit model; p < 0.05 spleen versus lung, liver, or kidney in CLP second hit model by one-way ANOVA). (F) Immunoprecipitation of U937 or THP1 cells transfected with vector encoding Flag-tagged Tim-3 or control vector and stimulated for 2 h with HMGB1 in the presence of control immunoglobulin (Ig) or mAb to Tim-3, followed by IP with mAb M2 to the FLAG tag and immunoblot analysis with anti-HMGB1 or anti-Flag. (G) Immunofluorescence image of Tim-3 (red) in Tim-3 + CD4 T cells colocalized with recombinant HMGB1 (green). Magnification, 600×.

Article Snippet: C57BL/6, Cd4 cre , Tim-3 fL/fL , and Tim-3 −/− mice were purchased from the Shanghai Model Organisms Center.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Binding Assay, Labeling, Immunoprecipitation, Transfection, Plasmid Preparation, Control, FLAG-tag, Western Blot, Immunofluorescence, Recombinant

Journal: Molecular Therapy

Article Title: Tim-3 regulates sepsis-induced immunosuppression by inhibiting the NF-κB signaling pathway in CD4 T cells

doi: 10.1016/j.ymthe.2021.12.013

Figure Lengend Snippet: Expression of Tim-3 on CD4 T cells regulates septic immunosuppression through the NF-κB/TNF-α pathway (A) Heatmap of the top 30 upregulated genes in Tim-3 – CD4 T cells compared to Tim-3 + CD4 T cells isolated from CLP second hit mice. (B) Enriched gene ontology functions of upregulated genes in Tim-3 – CD4 T cells. Upregulated genes were enriched in NF-κB signaling, LPS-stimulated MAPK, and Toll-like receptor signaling. (C) Western blot analysis of the indicated protein expression in Tim-3 + /Tim-3 – CD4 T cells in response to treatment with 100 ng/mL LPS for 0, 2, 4, or 6 h.

Article Snippet: C57BL/6, Cd4 cre , Tim-3 fL/fL , and Tim-3 −/− mice were purchased from the Shanghai Model Organisms Center.

Techniques: Expressing, Isolation, Western Blot