Journal: bioRxiv

Article Title: Eos promotes T H 2 differentiation by propagating the IL-2/STAT5 signaling pathway

doi: 10.1101/2022.11.02.514868

Figure Lengend Snippet: ( A ) Naïve WT CD4 + T cells from C57BL/6J mice were cultured on plates coated with α-CD3/α-CD28 stimulation under T H 2- and T FH -polarizing conditions for 3 days before being removed from stimulation and cultured in resting T H 2 (IL-4 and IL-2) and T FH (IL-6, IL-2) conditions for an additional 48 hours before harvesting on day 5. RNA was isolated and qRT-PCR was used to assess gene expression. Data were normalized to Rps18 (n = 4 independent experiments, mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001. **** P < 0.0001; one-way ANOVA with Tukey’s post-hoc test). (B) Cells were cultured as in ‘A’ under T REG -, T H 2-, T FH -, and T H 17-polarizing conditions. RNA was isolated and qRT-PCR was used to assess gene expression. Data were normalized to Rps18 . An immunoblot was also performed to assess the relative abundance of Eos (n = 4 independent experiments, mean ± s.e.m., *** P < 0.001. ****P < 0.0001; one-way ANOVA with Tukey’s post-hoc test). (C) Cells were cultured as in ‘A’ under T H 2-polarizing conditions only or with neutralizing antibodies for CD25 (α-IL-2Rα) and CD122 (α-IL-2Rβ). After 4 days, cells were harvested, RNA was isolated, and qRT-PCR was used to assess Ikzf4 (Eos) expression. Data were normalized to Rps18 and presented as fold change relative to anti-CD25/anti-CD122 sample. Immunoblot was performed to assess the relative abundance of Eos, pY-STAT5, and STAT5 expression (n = 4 independent experiments, mean ± s.e.m., ****P < 0.0001; unpaired Student’s t -test). (D) EL4 T cells were transfected with an Ikzf4 promoter-reporter construct in combination with either an empty vector control, constitutively active STAT5B (STAT5B CA ), STAT5B WT or STAT3 CA expression vectors. After 24 hours, cells were harvested for promoter-reporter assay. Luciferase promoter-reporter values were normalized to a Renilla control and presented relative to the empty vector control sample (n = 3 independent experiments, mean ± s.e.m., * P < 0.05, ** P < 0.01; one-way ANOVA with Tukey’s post-hoc test). (E) Chromatin Immunoprecipitation (ChIP) assays to assess STAT5 association with the Ikzf4 locus in in vitro T H 2 cells that were cultured, split at day 3, and harvested at day 5 as in ‘A’. The Ikzf4 promoter (“Prom”) and an upstream control region (“Ctrl”) were interrogated for STAT5 enrichment. Data are presented as percent enrichment relative to a “total” input sample. ChIP analysis was performed using anti-STAT5 and anti-IgG control antibodies for the indicated regions. Data are normalized to total input sample. IgG values were subtracted from percent enrichment ( n = 4 independent experiments, mean ± s.e.m., * P < 0.05; unpaired Student’s t -test).

Article Snippet: Resulting chromatin fragments were immunoprecipitated with antibodies against STAT5 (R&D AF2168, 10 μg/IP) or IgG control (Abcam ab6709; 10 μg/IP, matched to experimental antibody).

Techniques: Cell Culture, Isolation, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Construct, Plasmid Preparation, Reporter Assay, Luciferase, Chromatin Immunoprecipitation, In Vitro

Journal: bioRxiv

Article Title: Eos promotes T H 2 differentiation by propagating the IL-2/STAT5 signaling pathway

doi: 10.1101/2022.11.02.514868

Figure Lengend Snippet: (A) Co-Immunoprecipitation (Co-IP) of endogenously expressed proteins in T H 2 cells with control Ab (α-V5) or α-STAT5, followed by immunoblot with anti-Eos (n = 3 independent experiments). (B) Co-IP analysis of overexpressed V5-tagged Eos and tagless STAT5B CA in EL4 T cells. Lysates were immunoprecipitated with anti-STAT5, followed by immunoblot analysis with α-V5 (for detecting Eos) (n = 3 independent experiments). (C) Co-IP analysis of overexpressed Aiolos, Ikaros, or Eos with tagless STAT5B CA in EL4 T cells. Lysates were immunoprecipitated with α-STAT5, followed by immunoblot analysis with α-V5 (for detecting IkZF factors) (n = 3 independent experiments). (D) RNA was isolated from EL4 cells transfected with STAT5B CA , Eos, or STAT5B CA and Eos in combination. qRT-PCR was used to assess Il4ra expression. Data were normalized to Rps18 and presented as fold change relative to the empty vector sample (n = 4 independent experiments, mean ± s.e.m., **** P < 0.0001; one-way ANOVA with Tukey’s post-hoc test). (E) Immunoblot analysis of pY-STAT5, STAT5, and Eos protein expression in EL4 T cells transfected with STAT5B CA , Eos, or STAT5B CA and Eos in combination. β-actin serves as a loading control. Shown is a representative blot of 3 independent experiments. (F) Immunoblot analysis of pY-STAT5 and STAT5 in EL4 T cells transfected with tagless STAT5B CA alone or in combination with the indicated IkZF. β-actin serves as a loading control (n = 3 independent experiments).

Article Snippet: Resulting chromatin fragments were immunoprecipitated with antibodies against STAT5 (R&D AF2168, 10 μg/IP) or IgG control (Abcam ab6709; 10 μg/IP, matched to experimental antibody).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Isolation, Transfection, Quantitative RT-PCR, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Eos promotes T H 2 differentiation by propagating the IL-2/STAT5 signaling pathway

doi: 10.1101/2022.11.02.514868

Figure Lengend Snippet: (A) Heatmap of IL-2/STAT5 pathway DEGs both positively and negatively associated with the T H 2 gene program in WT vs. Eos-deficient cells; changes in gene expression are presented as relative expression by row (gene). (B,C) Pre-ranked (sign of fold change x −log 10 (p-value)) genes were analyzed using the Broad Institute GSEA software for comparison against ‘hallmark’ and ‘gene ontology’ gene sets. Data are compiled from 3 biological replicates from 3 independent experiments. (D) Immunoblot of day 3 WT vs. Eos-deficient T H 2 cells was performed to assess the relative abundance of pY-STAT5, STAT5, and Eos expression (n = 3 independent experiments).

Article Snippet: Resulting chromatin fragments were immunoprecipitated with antibodies against STAT5 (R&D AF2168, 10 μg/IP) or IgG control (Abcam ab6709; 10 μg/IP, matched to experimental antibody).

Techniques: Expressing, Software, Western Blot

Journal: bioRxiv

Article Title: Eos promotes T H 2 differentiation by propagating the IL-2/STAT5 signaling pathway

doi: 10.1101/2022.11.02.514868

Figure Lengend Snippet: (A) Deletion mutants of the conserved N-terminal DNA-binding (Eos ΔZF1,2 ) and C-terminal protein-protein interaction (Eos ΔC ) domains were created using the Agilent QuikChange Site-Directed Mutagenesis Kit. Wildtype and mutant coding sequences were transferred to the pEF1/V5-His vector for overexpression in EL4 T cells. (B) Co-IP of V5-tagged wild-type Eos (Eos WT ), Eos ΔC , and Eos ΔZF1,2 overexpressed with tagless STAT5B CA in EL4 T cells. Lysates were immunoprecipitated with anti-STAT5, followed by immunoblot analysis with α-V5 for detecting Eos proteins (n = 3 independent experiments). (C) Immunoblot analysis of pY-STAT5, STAT5, and Eos protein expression in EL4 T cells transfected with tagless STAT5B CA and either V5-tagged Eos WT , Eos ΔC , or Eos ΔZF1,2 . β-actin serves as a loading control (n = 3 independent experiments). (D) EL4 T cells were transfected with tagless STAT5B CA and either V5-tagged Eos WT , Eos ΔC , or Eos ΔZF1,2 . After 22h, RNA was isolated, and Il4ra expression was measured by qRT-PCR. Data were normalized to Rps18 as a control and presented as fold change in expression relative to Rps18 . (n = 4 independent experiments, mean ± s.e.m., ****P < 0.0001).

Article Snippet: Resulting chromatin fragments were immunoprecipitated with antibodies against STAT5 (R&D AF2168, 10 μg/IP) or IgG control (Abcam ab6709; 10 μg/IP, matched to experimental antibody).

Techniques: Binding Assay, Mutagenesis, Plasmid Preparation, Over Expression, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Expressing, Transfection, Isolation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Reliable Quantification of Protein Expression and Cellular Localization in Histological Sections

doi: 10.1371/journal.pone.0100822

Figure Lengend Snippet: A Western Blot of p-STAT5 Tyr694 expression in Stat5ab +/+ (n = 2), Stat5ab +/Δ (n = 2) and Stat5ab Δ/Δ (n = 2) livers of GH treated and control mice. HSC70 is shown as loading control. B IHC of p-STAT5 Tyr694 in sections of the livers (n = 3) used in A. Red, AEC; blue, hematoxylin. Size bar: 100 µm. High magnifications (60x) are shown in insets. C Quantification of p-STAT5 Tyr694 levels from Western blots (grey) by densitometric scanning and IHC (magenta) using the HistoQuest software ( Stat5ab +/+ , control; number of cells n = 3276, GH; number of cells n = 2286; Stat5ab +/Δ control; number of cells n = 2577, GH; number of cells n = 3549; Stat5ab Δ/Δ control; number of cells n = 3299, GH; number of cells n = 2200). The highest arbitrary expression values from A and B were set to 100 for direct comparison. D Western blot of p-STAT3 Tyr705 expression in Stat5ab +/+ (n = 2), Stat5ab +/Δ (n = 2) and Stat5ab Δ/Δ (n = 2) livers of GH treated and control mice. HSC70 is shown as loading control. E IHC of p-STAT3 Tyr705 in sections of the livers (n = 3) used in D. Red, AEC; blue, hematoxylin. Size bar: 100 µm. High magnifications are shown in insets (60x). F Quantification of p-STAT3 Tyr705 levels from Western blots (grey) by densitometric scanning and IHC (magenta) using the HistoQuest software ( Stat5ab +/+ , control; number of cells n = 2634, GH; number of cells n = 2286; Stat5ab +/Δ control; number of cells n = 22230, GH n = 2562; Stat5ab Δ/Δ control; number of cells n = 2414, GH n = 2716). The highest arbitrary expression values from A and B were set to 100 for direct comparison.

Article Snippet: Rabbit polyclonal JUNB (1∶300 dilution, sc-46, Santa Cruz) antibody, rabbit monoclonal p-STAT3 Tyr705 (1∶80 dilution, #9145, Cell Signaling), rabbit polyclonal STAT5AB (1∶200 dilution, sc-835, Santa Cruz) antibody and rabbit monoclonal p-STAT5AB Tyr694 (1∶80 dilution, #9314, Cell Signaling) antibody were used and incubated at 4°C overnight.

Techniques: Western Blot, Expressing, Control, Paraffin-embedded Immunohistochemistry, Software, Comparison

Journal: Molecular cell

Article Title: Cistromic Reprogramming of the Diurnal Glucocorticoid Hormone Response by High-Fat Diet

doi: 10.1016/j.molcel.2019.10.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sheared chromatin was then immunoprecipitated with α-GR antibody (Santa Cruz, sc-1004X; Proteintech, 24050–1-AP), α-PPARa antibody (Santa Cruz, sc-9000), α-STAT5a/b (RD System, AF2168; Cell Signaling, 94205; Abcam, ab194898), α-STAT5a/b (pY694/Y699) (Cell Signaling, 9351) or α-H3K27ac (Abcam, ab4729), and DNA was isolated with MinElute PCR Purification Kits (QIAGEN).

Techniques: Recombinant, Lysis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software