Journal: The Journal of Experimental Medicine

Article Title: Arid5a regulates naive CD4 + T cell fate through selective stabilization of Stat3 mRNA

doi: 10.1084/jem.20151289

Figure Lengend Snippet: Selective stabilization of Stat3 mRNA by Arid5a in CD4 + T cells under Th17-polarizing conditions. (A) Quantitative real-time PCR analysis of mRNAs of Stat1 , Stat3 , Stat5a , and Rorc in CD4 + T cells differentiated for 12 h under Th17 cell-inducing conditions, followed by treatment for 0–180 min with actinomycin D (ActD). (B) Luciferase activity of HEK 293T cells transfected for 48 h with the luciferase reporter vector (100 ng) encoding the STAT3 human promoter in combination with Arid5a expression vector (100–300 ng) or empty vector (100–300 ng). (C) Luciferase activity of HEK 293T cells transfected for 48 h with pGL3 vector encoding the total STAT3 3′UTR, IL-2 3′UTR, IL-17 3′UTR, or RORc 3′UTR in combination with Arid5a expression vector, empty vector, or Arid5a mutant (lacking Arid domain) vector. (D) Immunoblot analysis of Arid5a expression in HEK293T cells transfected for 48 h with Flag-Arid5a expression vector or empty vector (EV). (E) Quantitative real-time PCR analysis of mRNAs of Stat3 and Arid5a in either WT or Arid5a-deficient CD4 + T cells differentiated for 0, 1, 2, 6, 12, 24, 48, or 72 h under Th17 cell conditions. Time-course expression (0–6 h) of Stat3 and Arid5a in WT or Arid5a-deficient CD4 + T cells was enlarged (right). Data are representative of two independent experiments with similar results. (F) Quantitative real-time PCR analysis of mRNAs of Stat3 , Batf , Rorc , or Rora in either WT or Arid5a-deficient CD4 + T cells differentiated for 48 h under Th17 cell conditions. Data are representative of three independent experiments (A–D and F). Error bars show mean ± SD (A–C and F). *, P < 0.05; **, P < 0.02; ***, P < 0.001 (Student’s t test).

Article Snippet: HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector.

Techniques: Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Arid5a regulates naive CD4 + T cell fate through selective stabilization of Stat3 mRNA

doi: 10.1084/jem.20151289

Figure Lengend Snippet: The STAT3 3′UTR (1449–1792) is the responsive sites of Arid5a and an endoribonuclease Regnase-1. (A) Diagram of the region of pGL3 vector encoding STAT3 3′UTR (1–1895), STAT3 3′UTR (1–901), or STAT3 3′UTR (902–1895). The black bar shows AU-rich elements (ARE). (B) Luciferase activity of the vector encoding the STAT3 3′UTR (1–1895) in HEK293T cells transfected for 48 h together with Arid5a expression vector, Regnase-1 expression vector, or empty vector. (C) Quantitative real-time PCR analysis of mRNAs of Regnase-1 ( Reg-1 ) and Stat3 in Jurkat cells electroporated with Reg-1 siRNA or control siRNA, normalized to human Gapdh mRNA expression. (D) Quantitative real-time PCR analysis of Regnase-1 ( Reg-1 ) mRNA in Regnase-1–deficient MEFs, normalized to Gapdh mRNA expression. (E) Luciferase activity of the pGL3-vector encoding the STAT3 3′UTR (1–901) or STAT3 3′UTR (902–1895), or the pGL3-empty vector cotransfected for 48 h with Arid5a expression vector. (F) Diagram of the region of pGL3 vector encoding the STAT3 3′UTR (902–1458), STAT3 3′UTR (1449–1792), STAT3 3′UTR (1698–1895), or STAT3 3′UTR (1773–1895). The black bar shows AU-rich elements (ARE). (G) Luciferase activity of the vector encoding each area of the STAT3 3′UTR as in F cotransfected with either Arid5a expression vector or Regnase-1 expression vector. (H) Luciferase activity of the vector encoding the total STAT3 3′UTR (1–1895) in HEK293T cells for 48 h cotransfected with either Regnase-1 vector or Arid5a expression vector. Data are representative of three independent experiments (B–E, G, and H). Error bars show mean ± SD (B–E, G, and H). *, P < 0.05; **, P < 0.02; ***, P < 0.001 (Student’s t test).

Article Snippet: HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector.

Techniques: Plasmid Preparation, Luciferase, Activity Assay, Transfection, Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: Arid5a regulates naive CD4 + T cell fate through selective stabilization of Stat3 mRNA

doi: 10.1084/jem.20151289

Figure Lengend Snippet: Arid5a physically associates with the stem structure of the STAT3 3′UTR (1738–1765) via its residue R128. (A) A predicted conserved stem-loop region in the STAT3 3′UTR between human and mouse. Blue and red colored regions indicate stem and loop regions, respectively. (B and C) Secondary structure models of the stem-loop regions of mouse (B) and human (C) as in A. The conserved elements between human and mice are highlighted in blue. (D) Identification of the secondary structure of the STAT3 3′UTR (1738–1765) by NMR analysis. The trace of the NOESY connectivity is indicated by bold solid lines. Closed circles indicate base pairs that were experimentally identified. (E) Diagram of the stem-loop structure (1738–1765) of the STAT3 3′UTR and recombinant mouse Arid5a proteins. (F) Gel mobility shift assay (EMSA) of interaction of Arid5a recombinant protein with 3′-biotinated nucleotides as in E. (G) Docking model of Arid5a-STAT3 3′UTR (1738–1765) in silico. (H) EMSA of noninteraction of mutant Arid5a recombinant protein or interaction of Arid5a recombinant protein with 3′-biotinated nucleotides as in E. (I) Immunoblot analysis of recombinant Arid5a protein or mutant recombinant Arid5a by anti-Arid5a antibody, respectively. (J) Diagram of the stem-loop structure of the STAT3 3′UTR (1738–1765) and the mutant stem-loop structure. (K) EMSA of noninteraction of the 3′-biotinated mutant stem-loop or interaction of the 3′-biotinated stem-loop with Arid5a recombinant protein as in J. Data are representative of three independent experiments (D, F, H, I, and K).

Article Snippet: HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector.

Techniques: Recombinant, Mobility Shift, In Silico, Mutagenesis, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Arid5a regulates naive CD4 + T cell fate through selective stabilization of Stat3 mRNA

doi: 10.1084/jem.20151289

Figure Lengend Snippet: Arid5a inhibits the binding of Regnase-1 to the stem-loop STAT3 3′UTR (1738–1765). (A) Schematic diagram of an RNA-protein binding assay. The lysates were prepared from HEK293T cells transfected with Flag-Regnase-1 expression vector or empty vector for 48 h. The 3′-biotinated nucleotides (5′-UGCAGUGGCUUGUGUUCUGGCCACUGCA-3′) of the STAT3 3′UTR (1738–1765) was conjugated with Streptavidin beads. Next, the lysates and the beads were mixed, and then washed three times. Finally, the binding proteins with the beads were eluted. (B) Immunoblot analysis of Flag-Regnase-1 protein in HEK293T cells for 48 h transfected with Flag-Regnase-1 expression vector or Flag-Regnase-1 protein binding to the stem-loop STAT3 3′UTR (1738–1765). (C) Schematic diagram of a competitive assay of RNA-protein binding. The lysates with or without Arid5a recombinant protein were prepared from HEK293T cells transfected with Flag-Regnase-1 expression vector for 48 h. Next, the lysates were mixed with the RNA-binding beads as in A, and washed three times. Finally, the binding proteins were eluted. (D) Immunoblot analysis of recombinant Arid5a protein and Flag-Regnase-1 protein in the lysates or the eluted samples, as in C. Data are representative of three independent experiments (B and D).

Article Snippet: HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector.

Techniques: Binding Assay, Protein Binding, Transfection, Expressing, Plasmid Preparation, Western Blot, Recombinant, RNA Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Arid5a regulates naive CD4 + T cell fate through selective stabilization of Stat3 mRNA

doi: 10.1084/jem.20151289

Figure Lengend Snippet: Reduction of STAT3 level in T cells lacking Arid5a impairs a balance among STAT1, STAT3, and STAT5 activation under Th17-polarizing conditions. (A) Immunoblot analysis of STAT3, phosphorylated STAT3, and β-actin in WT or Arid5a-deficient CD4 + T cells differentiated for the indicated times toward Th17 cells. (B) Intensity of STAT3 or phosphorylated STAT3 level as in A. (C) Immunoblot analysis of STAT1, phosphorylated STAT1, and β-actin in WT or Arid5a-deficient CD4 + T cells differentiated for the indicated times toward Th17 cells. (D) Intensity of STAT1 or phosphorylated STAT1 level as in C, respectively. (E and F) Phosphorylation of STAT3 or STAT1 in WT or Arid5a-deficient CD4 + T cells for 30 min differentiated toward Th17 cells by FACS analysis. (G and H) Immunoblot analysis of Arid5a or Regnase-1 protein in CD4 + T cells differentiated for the indicated times into Th17 cells. (I) Immunoblot analysis of STAT5, phosphorylated STAT5, and β-actin in WT or Arid5a-deficient CD4 + T cells differentiated for the indicated times toward Th17 cells. (J) Intensity of STAT5 or phosphorylated STAT5 level as in I. Data are representative of three independent experiments (A–J). Data are representative from three independent experiments with similar results (B, D, and J).

Article Snippet: HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector.

Techniques: Activation Assay, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Arid5a regulates naive CD4 + T cell fate through selective stabilization of Stat3 mRNA

doi: 10.1084/jem.20151289

Figure Lengend Snippet: Decreased STAT3 level in Arid5a-deficient T cells critically contributes to the attenuation of Th17 cell populations. (A) Immunoblot analysis of STAT3 and β-actin in WT or Arid5a-deficient CD4 + T cells for 48 h stimulated by anti-CD3ε and anti-CD28 antibodies with or without IL-6 plus TGF-β or IL-6 alone. (B and C) WT and Arid5a-deficient CD4 + T cells were transfected with MIG or MIG-STAT3C retrovirus vector for 24 h, respectively, and then cultured for 4 d under Th17-polarizing conditions. STAT3 or β-actin level in these cells was analyzed by immunoblot analysis, respectively (B). The frequency of Th17 cells in WT or Arid5a-deficient T cells by FACS analysis (C). Error bars show mean ± SD (C). (D) FACS analysis of WT or Arid5a-deficient T cells differentiated for 5 d under Th17-polarizing conditions with anti-IL-2 (JES6-1A12) treatment or isotype control (IC). (E) FACS analysis of CD4 + WT or Arid5a-deficient T cells differentiated for 5 d into Th17 cells after the stimulation of anti-CD3ε and anti-CD28 antibodies with IL-6 and IL-23 plus TGF-β (left) or IL-1β (right). Data are representative of three independent experiments (A–E).

Article Snippet: HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector.

Techniques: Western Blot, Transfection, Plasmid Preparation, Cell Culture