Journal: JCI Insight

Article Title: Aiolos promotes CXCR3 expression on Th1 cells via positive regulation of IFN- γ /STAT1 signaling

doi: 10.1172/jci.insight.180287

Figure Lengend Snippet: ( A ) Publicly available chromatin immunoprecipitation sequencing (ChIP-Seq) data for STAT1 (GSM994528), STAT4 (GSM550303), and T-bet (GSM836124) were examined at Cxcr3 . Sequencing tracks were viewed using the Integrative Genomics Viewer (IGV). Regulatory regions of interest with transcription factor enrichment are indicated by the blue boxes. ( B ) Published RNA-Seq data (GSE203065) from in vitro–generated WT and Ikzf3 –/– Th1 cells were analyzed for DEGs. A heatmap of DEGs associated with IFN-γ/STAT1 and IL-12/STAT4 signaling in Th1 cells is shown, as well as additional genes involved in both pathways and Th cell differentiation. Gene names color-coded in blue are downregulated in Ikzf3 –/– Th1 cells. Note: Cxcr3 transcript data presented here are the same as in . ( C ) Schematic of proposed model in which Aiolos may regulate CXCR3 via impacts on components of the IFN-γ/STAT1 and IL-12/STAT4 cytokine signaling pathways. The downward arrows in blue indicate genes that are downregulated in the absence of Aiolos.

Article Snippet: The following antibodies were used to detect proteins: α-JAK2 (1:1,000; 3230, Cell Signaling Technology), α–pY-STAT4 (1:1,000; 5267, Cell Signaling Technology), α-STAT4 (1:1,000; 2653S, Cell Signaling Technology), α–pY-STAT1 (1:1,000; 9167S, Cell Signaling Technology), α-STAT1 (1:1,000; sc-417, Santa Cruz Biotechnology), α-Aiolos (1:20,000; 39293, Active Motif), α–β-actin–HRP (1:15,000; A00730, GenScript), goat α-mouse (1:5,000; 115-035-174, Jackson Immunoresearch), and mouse α-rabbit (1:5,000–1:10,000; sc-2357, Santa Cruz Biotechnology).

Techniques: ChIP-sequencing, Sequencing, RNA Sequencing, In Vitro, Generated, Cell Differentiation, Protein-Protein interactions

Journal: Cell Reports

Article Title: CDK8-Mediated STAT1-S727 Phosphorylation Restrains NK Cell Cytotoxicity and Tumor Surveillance

doi: 10.1016/j.celrep.2013.07.012

Figure Lengend Snippet: Constitutive Basal STAT1-S727 Phosphorylation in NK Cells Is Mediated by CDK8 (A) Western blot of freshly isolated DX5 + splenocytes from wild-type animals shows constitutive basal phosphorylation on STAT1-S727 in vivo. (B and C) In vitro-expanded LAK cells from wild-type and Stat1-S727A mice were treated with or without (B) 100 U/ml IFN-β or (C) 5 ng/ml IL-12 for the given times, and the phosphorylation on STAT1-Y701 and STAT1-S727 was investigated by western blot. Stat1 −/− LAK cells served as negative control and HSC-70 as loading control. (D) Wild-type LAK cells were treated with different kinase inhibitors targeting CDKs (roscovitine and flavopiridol) or MAPK p38 (BIRB 0796 and SB 203580), and the phosphorylation status of STAT1-S727 was determined in a western blot. (E) CDK8 knockdown in primary NK cells with two hairpins led to reduced STAT1-S727 phosphorylation. A random hairpin served as control. (F) Knockdown of CDK8 with five hairpins in an immortalized Ink4a −/− NK cell line is shown. A random hairpin served as control. (G) FACS-based cytotoxicity assay of immortalized NK cells transduced with a random hairpin or a hairpin against CDK8 (#3) against YAC-1 target cells is presented. Symbols represent mean, and error bars indicate SEM of triplicates ( ∗ p < 0.05, ∗∗∗ p < 0.001; unpaired t test). See also Figure S2 .

Article Snippet: After blocking with 5% BSA in pY-TBST buffer (10 mM Tris/HCl pH 7.4, 75 mM NaCl, 1 mM EDTA, 0.1% Tween-20), membranes were probed with antibodies against α-tubulin (sc-32293), HSC-70 (sc-7298) and STAT1 (sc-592) (Santa Cruz Technology), CDK8 (CS#4101), pSTAT1-Y701 (CS#9171), pSTAT1-S727 (CS#9177), STAT3 (CS#9132), pSTAT3-Y705 (CS#9131), STAT4 (CS#2653) (Cell Signaling) and pSTAT4-Y693 (BD 612738, BD Biosciences).

Techniques: Phospho-proteomics, Western Blot, Isolation, In Vivo, In Vitro, Negative Control, Control, Knockdown, Cytotoxicity Assay, Transduction