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: 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 ) Schematic of culturing system. Naive CD4 + T cells were stimulated with α-CD3/CD28 and cultured under Th1-polarizing conditions (IL-12, α–IL-4). On day 3, cells were removed from stimulation and given IFN-γ, α–IL-4, and IL-2 for an additional 2 days. ( B ) At day 5, transcript analysis was performed via qRT-PCR. Transcript was normalized to Rps18 and presented as fold-change compared with WT control ( n = 4 biological replicates from 4 independent experiments, mean ± SEM; ** P < 0.01, *** P < 0.001, **** P < 0.0001, 2-tailed unpaired Student’s t test). ( C ) Representative flow cytometric analysis for CXCR3 on IFN-γ–treated Th1 cells at day 5. Data are displayed as MFI fold-change compared with WT controls ( n = 3 biological replicates from 3 independent experiments, mean ± SEM; ** P < 0.01, 2-tailed unpaired Student’s t test). ( D ) An immunoblot was performed to assess the relative abundance of the indicated proteins. β-Actin serves as a loading control ( n = 4 independent experiments, mean ± SEM; * P < 0.05, *** P < 0.001, 2-tailed unpaired Student’s t test). ( E ) ChIP assays were performed to assess STAT1 association with Cxcr3 in WT and Ikzf3 –/– Th1 cells. Publicly available ChIP-Seq data for STAT1 (GSM994528) were examined to identify potential regions of STAT1 enrichment. Sequencing tracks were viewed using IGV, and regulatory regions of interest are indicated by blue boxes. Approximate ChIP primer locations at the Cxcr3 promoter (prom.) and 3′ enhancer (enhc.) are indicated with gray arrows. ( F ) The indicated regions were analyzed for STAT1 enrichment. Data were normalized to total input. Percentage enrichment relative to input was divided by IgG, and data are presented as fold-change relative to IgG. ( n = 4 biological replicates from 4 independent experiments, mean ± SEM; ** P < 0.01, *** P < 0.001, 1-way ANOVA with Tukey’s multiple comparisons test.)

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: Cell Culture, Quantitative RT-PCR, Control, Western Blot, ChIP-sequencing, Sequencing

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 ) Schematic of culturing system. WT naive CD4 + T cells were stimulated with α-CD3/CD28 under Th1-polarizing conditions (IL-12, α–IL-4). Some cells were also treated with α–IFN-γ to inhibit IFN-γ/STAT1 signaling. ( B ) At day 3, transcript analysis was performed via qRT-PCR. Transcript was normalized to Rps18 and presented as fold-change compared with WT control ( n = 4 biological replicates from 4 independent experiments, mean ± SEM; ** P < 0.01, **** P < 0.0001, 2-tailed unpaired Student’s t test). ( C ) Representative flow cytometric analysis at day 3 for CXCR3 expression on WT Th1 cells treated with or without α–IFN-γ. Data are displayed as percentage positive for CXCR3 ( n = 3 biological replicates from 3 independent experiments, mean ± SEM; * P < 0.05, 2-tailed unpaired Student’s t test). ( D ) An immunoblot was performed to assess the relative abundance of the indicated proteins. β-Actin serves as a loading control ( n = 4 independent experiments, mean ± SEM; * P < 0.05, ** P < 0.01, **** P < 0.0001, 2-tailed unpaired Student’s t test). ( E ) At day 3, transcript and flow cytometric analyses were performed for Ikzf3 and Aiolos protein expression, respectively. Flow cytometric data are displayed as MFI fold-change compared with WT controls ( n = 3 biological replicates from 3 independent experiments, mean ± SEM; ** P < 0.01, 2-tailed unpaired Student’s t test).

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: Quantitative RT-PCR, Control, Expressing, Western Blot

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 ATAC-Seq data (GSE203064) from WT and Ikzf3 –/– Th1 cells were assessed for alterations in chromatin accessibility at the Stat1 promoter. Publicly available ChIP-Seq data for Aiolos (GSM5106065) were examined at Stat1 . Sequencing tracks were viewed using IGV. The Stat1 promoter region of significant differential accessibility is indicated by a blue box ( P adj = 0.0302). A ~500 bp region encompassing the indicated Aiolos DNA binding motifs within the Stat1 promoter was subcloned into a reporter plasmid. ( B ) Schematic depicting the zinc finger (ZF) domains of WT Aiolos and a DNA-binding mutant (Aiolos DBM ). ( C ) EL4 T cells were transfected with a Stat1 promoter-reporter and WT Aiolos, Aiolos DBM , or empty vector control. Cells were also transfected with SV40- Renilla as a control for transduction efficiency. Luciferase promoter-reporter values were normalized to Renilla control and presented relative to the empty vector control. Aiolos was assessed via immunoblot with an antibody for the V5 epitope tag. β-Actin serves as a loading control. Data are representative of 3 independent experiments ( n = 3, mean ± SEM; * P < 0.05, 1-way ANOVA with Tukey’s multiple comparisons test). ( D ) Publicly available ATAC-Seq data (GSE203064) from Th1 cells and ChIP-Seq data for STAT1 (GSM994528) were viewed using IGV to identify regions of STAT1 enrichment (blue box) at Ikzf3 . Approximate ChIP primer locations are indicated with a gray arrow. ( E ) The Ikzf3 promoter (prom.) and a negative control region (neg. ctrl.) were analyzed for STAT1 enrichment via ChIP. Data were normalized to total input. Percentage enrichment relative to input was divided by IgG, and data are presented as fold-change relative to IgG. ( n = 4 biological replicates from 4 independent experiments, mean ± SEM; * P < 0.05, ** P < 0.01, 1-way ANOVA with Tukey’s multiple comparisons test.)

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, Binding Assay, Plasmid Preparation, Mutagenesis, Transfection, Control, Transduction, Luciferase, Western Blot, Negative Control

Journal: Genesis (New York, N.Y. : 2000)

Article Title: Generation of Janus kinase 1 (JAK1) conditional knockout mice

doi: 10.1002/dvg.22982

Figure Lengend Snippet: Conditional deletion of Jak1 in the female germline and consequential effects of JAK1 deficiency on STAT activation and embryonic development. (a) Breeding strategy to convert the floxed allele of Jak1 (Jak1fl) into a null mutation (Jak1−). MMTV-Cre (line A) mice express Cre recombinase in developing oocytes. Thus, heterozygous Jak1fl/wt MMTV-Cre females transmit a null allele to their offspring whether the Cre transgene is segregated out or not. (b) PCR assays using the primer sets illustrated in panel Figure 1a,​,cc to validate the germline deletion of the Jak1 floxed allele and the presence of the recombined knockout/null allele of Jak1. An internal primer set for neo served as a positive control for the presence of the mutant alleles, and the amplification of a PCR fragment of the Jak2 gene was used to control for the integrity of the DNA samples. (c) Immunoblot analyses of the expression of JAKs 1 and 2 as well as the tyrosine phosphorylation of STATs 1, 3, and 6 in immortalized mouse embryonic fibroblasts (MEFs) of heterozygous and homozygous JAK1 knockouts (Jak1wt/−, Jak1−/−) as well as wildtype controls (Jak1wt/wt). Beta-actin (ACTB) was used as a loading control. Immunoprecipitation (IP)/western blot analyses was used to determine the phosphorylation on tyrosine 705 and serine 727 on STAT3. (d) Comparison of JAK1 deficient embryos and littermate wildtype controls during the final stages of prenatal development. A subset of JAK1 knockout pups were alive at birth but showed symptoms of apnea. None of the pups were able to survive

Article Snippet: The following antibodies were used for immunoblotting: α-β-actin (I-19), α-STAT1 (sc-592), and α-STAT6 (sc-981) from Santa Cruz Biotechnology; α-STAT3 (9139S), α-pY-STAT3(Y705) (9145S), and α-pS-STAT3(S727) (9136S) from Cell Signaling, Inc., α-pY-STAT6(Y641) (ab54461) from Abcam, α-pY-STAT1(Y701) (TA309955) from Origene.

Techniques: Activation Assay, Mutagenesis, Knock-Out, Positive Control, Amplification, Control, Western Blot, Expressing, Phospho-proteomics, Immunoprecipitation, Comparison

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