Journal: bioRxiv

Article Title: Selective epigenetic regulation of IFN-γ signature genes by JAK inhibitor in inflammatory diseases

doi: 10.1101/2024.08.05.606293

Figure Lengend Snippet: (A) Flow cytometry assessment of STAT1 and STAT3 protein levels in THP-1 cells treated with PMA and IFN-γ (or without) at various time points. Kinetics of total STAT and phospho-STAT tyrosine (STAT1, Tyr701; STAT3, Tyr705) measured by flow cytometry in IFN-γ-primed macrophages compared to resting macrophages. (B) Kinetics of total STAT1 (or STAT3) and phospho-STAT1 (or STAT3) measured by flow cytometry in JAK inhibitor-treated macrophages compared to resting and JAK inhibitor-untreated macrophages. Resting and IFN-γ-primed macrophages were differentiated for 24 h and then treated with JAKi (or DMSO) for up to 6 h. (A and B) Mean fluorescence intensity (MFI) represents a fold change compared to unstained samples. (C) RT-qPCR analysis of normalized target mRNA relative to TBP mRNA in THP-1 monocyte-derived macrophages under indicated conditions. IFN-γ-primed macrophages were treated with JAK inhibitor at a concentration of 1 μM for up to 6 h. Data show means ± SD from two independent experiments. (D) K-means clustering of differentially expressed (DE) genes in pairwise comparisons between the four conditions. DE genes identified by EdgeR (FDR adjusted P < 0.05, fold change > 2) were used. TPM values of RNA-seq data were filtered to be greater than 4. Non-significant clusters between replications were removed, resulting in three identified clusters. Clusters are indicated on the left. (E-G) Examples of expression for selected genes from clusters identified in the heatmap. Each dot on the bar plot represents one sample, and error bars denote the standard deviation. Error bars represent means ± SD. (H) Gene ontology (GO) analysis of THP-1 RNA-seq using genes positively correlated with HMDM RNA-seq. Heatmap displays the P-value (-Log10) significance of GO term enrichment for genes in each cluster, with clusters shown at the top. Downregulated by IFN-γ, n = 42; JAKi-sensitive, n = 129; JAKi-insensitive, n = 112. (I) Identification of genes associated with JAKi-sensitive or JAKi-insensitive in RA patients. Clusters are indicated on the left. (J) Identification of genes associated with JAKi-sensitive or JAKi-insensitive in COVID-19 patients. Clusters are indicated on the left. For heatmaps of single cells, mean expression values were used, and hierarchical analysis was performed. (K) GO analysis of overlapping JAKi-sensitive and JAKi-insensitive genes in RA or COVID-19 patients. Clusters are indicated on the left. JAKi-sensitive, n = 51; JAKi-insensitive, n = 24. p < 0.05(*), p < 0.01(**), p < 0.001(***) and p < 0.0001(****) by one-way ANOVA. GO analysis was performed using Metascape ( http://metascape.org/ ).

Article Snippet: Add antibody (Alexa Fluor® 647 Mouse anti-Total Stat1, BD biosciences, 58560; PE anti-STAT1 Phospho (Tyr701), BioLegend, 666404; APC Mouse anti-Total Stat3, BD biosciences, 560392; PE/Cyanine5 anti-STAT3 Phospho (Tyr705), BioLegend, 651014) cocktail(s) to appropriate tubes, vortex to mix, and incubate for 30 minutes at room temperature in the dark.

Techniques: Flow Cytometry, Fluorescence, Quantitative RT-PCR, Derivative Assay, Concentration Assay, RNA Sequencing Assay, Expressing, Standard Deviation

Journal: iScience

Article Title: Interferon hyperactivity impairs cardiogenesis in Down syndrome via downregulation of canonical Wnt signaling

doi: 10.1016/j.isci.2023.107012

Figure Lengend Snippet: Trisomy 21 causes hyperactivation of IFN signaling and impairs cardiac differentiation in vitro (A) Representative control (C68) and DS/CHD (D49) Ca 2+ transient traces measured with overexpressed GCaMP6f in iPSC-CMs on day 13 of cardiac differentiation. Florescence intensity was normalized to baseline (F/F 0 ). Quantification of Ca 2+ transient amplitude and frequency is shown in Figure S3 . (B) Heatmap of gene expression as assayed by RNA-seq in control and DS/CHD cells on differentiation day 7. Two replicates (rep1 and rep2) were used for each iPSC line for RNA-seq. (C) Top GO terms associated with significantly up- and downregulated genes with a >1.5-fold change in DS/CHD cells on differentiation day 7, compared with control. (D) Heatmap of gene expression as assayed by RNA-seq in control and DS/CHD cells on differentiation day 13. Two replicates (rep1 and rep2) were used for each iPSC line for RNA-seq. (E) GO analysis of significantly up- and downregulated genes with a >1.5-fold change in DS/CHD cells on differentiation day 13, compared with control. (F) Gene set enrichment analysis (GSEA) of RNA-seq on day 0 and day 3 showing that genes upregulated in cells differentiated from DS/CHD iPSCs were enriched in IFN gamma signaling response. RNA-seq was performed on C62, C68, D7, and D49 iPSC lines. Genes with expression ≥25 FPKM were selected for analysis. Genes preferentially expressed in DS/CHD cells are shown in Figures S4 A and S4B. (G and H) Immunoblotting analysis of phosphorylated STAT1(Tyr701) (p-STAT1) and GAPDH in cells differentiated from control (Ctrl) (C62 and C68) and DS/CHD (D7 and D49) iPSCs on differentiation day 3. Representative western blots are shown in G. Quantification of relative p-STAT1 levels are presented in H. Each filled circle represents one independent experiments for each iPSC line. Three independent inductions were performed for each line. Data are presented as mean ± SD. ∗∗∗p < 0.001, unpaired Student’s t test.

Article Snippet: The primary antibodies include anti-active β-Catenin (Millipore, 1:1000), anti-total β-Catenin (Cell signaling, 1:1000), anti-ISL1 (Developmental Studies Hybridoma Bank, 1:1000), anti-Phospho-STAT1 (Tyr701) (Cell signaling, 1:1000), anti-total STAT1 (Cell signaling, 1:1000), anti-GATA4 (Santa Cruz, 1:500), and anti-GAPDH (Ambion,1:5,000).

Techniques: In Vitro, Control, Gene Expression, RNA Sequencing, Expressing, Western Blot

Journal: iScience

Article Title: Interferon hyperactivity impairs cardiogenesis in Down syndrome via downregulation of canonical Wnt signaling

doi: 10.1016/j.isci.2023.107012

Figure Lengend Snippet: Reduction of the activity of IFN signaling pathways reverses defects in cardiac differentiation of DS/CHD iPSCs (A and B) Immunoblotting analysis of p-STAT1 and GAPDH on differentiation day 3. DS/CHD iPSCs were treated with DMSO or 1 μM JAKi from day 0 to day 3. A representative immunoblotting image is shown in A. Quantification of relative p-STAT1 levels in control cells (C62 and C68) and DS/CHD cells (D7 and D48) on differentiation day 3 in B. Each filled circle represents one independent induction, with two independent experiments for each line. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ns, not significant, ordinary one-way ANOVA. (C) qPCR analysis of gene expression in iPSC-derived cells on differentiation day 7. DS/CHD iPSCs were treated with 1.0 μM JAKi beginning on day 0. Each filled circle represents one independent induction, with three independent inductions for each cell line. Control lines are C62 and C68 and DS/CHD lines are D7 and D49. Data are presented as mean ± SD. ∗p < 0.05, Ordinary one-way ANOVA. (D) Representative traces of normalized GCaMP6f signal intensity showing Ca 2+ transients in control iPSC-CMs (C68), DS/CHD iPSC-CMs (D49), and DS/CHD iPSC-CMs treated with the JAKi from day 0 to day 3. Ca 2+ signals were imaged on day 13. Merged traces in the box are shown on right. Quantification of Ca 2+ transient amplitude and frequency is shown in Figure S3 . (E and F) Transcriptomic analysis using RNA-seq. Three independent experiments were performed for each iPSC line (C62, C68, D7, and D48) under each condition for RNA-seq. FPKM ≥30 was set as cutoff to filter transcripts. Fold change ≥1.5 and p < 0.05 were set as criteria to determine DEGs. Venn diagrams in E demonstrating numbers of genes whose dysregulation was rescued by upon JAKi treatment on differentiation day 13. GO analysis in F of the overlapping downregulated and upregulated DEGs in DS/CHD-differentiated cells shown in E on day 13 and the rescue effects of the JAKi.

Article Snippet: The primary antibodies include anti-active β-Catenin (Millipore, 1:1000), anti-total β-Catenin (Cell signaling, 1:1000), anti-ISL1 (Developmental Studies Hybridoma Bank, 1:1000), anti-Phospho-STAT1 (Tyr701) (Cell signaling, 1:1000), anti-total STAT1 (Cell signaling, 1:1000), anti-GATA4 (Santa Cruz, 1:500), and anti-GAPDH (Ambion,1:5,000).

Techniques: Activity Assay, Protein-Protein interactions, Western Blot, Control, Gene Expression, Derivative Assay, RNA Sequencing

Journal: iScience

Article Title: Interferon hyperactivity impairs cardiogenesis in Down syndrome via downregulation of canonical Wnt signaling

doi: 10.1016/j.isci.2023.107012

Figure Lengend Snippet: Pharmacological inhibition of IFN signaling prevents heart malformations in Dp16 embryos (A and B) Immunoblotting analysis of heart extracts from indicated embryos at E15.5 for p-STAT1, total STAT1, and GAPDH. Representative western blots are shown in A. Quantification of protein levels from western blots are in B. Each filled circle represents one individual embryonic heart. Data are presented as mean ± SD. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ordinary one-way ANOVA. (C and D) Immunoblotting analysis of heart extracts from indicated embryos at E15.5 for p-STAT1 and GAPDH. Pregnant mice were daily treated with the JAKi (10 mg/kg body weight/day, i.p. injection) beginning on day 6.5 post-conception. Hearts were harvested at E15.5 for analysis. Protein quantification is shown in D. Each filled circle represents one individual heart. Data are presented as mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001, Ordinary one-way ANOVA. (E and F) Histological analysis of cardiac septation in embryos at E15.5. Representative images of hematoxylin & eosin-stained sections of embryos are shown in E. Normal septation of four chambers in a WT embryo, and atrial septal defect (ASD) or ventricular septal defect (VSD), which were each observed in Dp16 embryos. LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle. Percentages of E15.5 embryos displaying heart malformations are shown in F. Scale bar, 300 μm. ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant, Fisher’s exact test. (G) GO analysis of the dysregulated DEGs, as assessed by RNA-seq, associated with variable IFN signaling and JAKi treatment. FPKM ≥30 in WT samples was set as cutoff to filter transcripts. DEGs were determined by Student’s t test (p < 0.05). N = 3–4 hearts for each group. (H and I) Examination of cardiac troponin I (cTnI) and active β-Catenin levels in embryos at E9.5 by immunofluorescence (IF) analysis. Pregnant mice were treated daily with vehicle or the JAKi (10 mg/kg body weight/day, i.p.) beginning with day 6.5 post-conception. Embryos were harvested at E9.5 for IF analysis. Representative IF images are presented in H. Scale bars on top panels, 400 μm. Scale bars on middle and bottom panels, 50 μm. Each white box indicates area of magnification in bottom images. Quantification of IF signaling intensity for active β-Catenin in cTnI + developing hearts in I. Two sections were analyzed for each embryo. Each filled circle represents the average of active β-Catenin signals in one embryo. ∗∗p < 0.05, ∗∗∗p < 0.05, ns, not significant, ordinary one-way ANOVA. (J) T21 leads to increased expression of IFN receptors encoded by genes on HSA21, which overactivates IFN signaling. Activated IFN signaling decreases the activity of the canonical Wnt pathway, eventually leading to defects during heart development.

Article Snippet: The primary antibodies include anti-active β-Catenin (Millipore, 1:1000), anti-total β-Catenin (Cell signaling, 1:1000), anti-ISL1 (Developmental Studies Hybridoma Bank, 1:1000), anti-Phospho-STAT1 (Tyr701) (Cell signaling, 1:1000), anti-total STAT1 (Cell signaling, 1:1000), anti-GATA4 (Santa Cruz, 1:500), and anti-GAPDH (Ambion,1:5,000).

Techniques: Inhibition, Western Blot, Injection, Staining, RNA Sequencing, Immunofluorescence, Expressing, Activity Assay

Journal: iScience

Article Title: Interferon hyperactivity impairs cardiogenesis in Down syndrome via downregulation of canonical Wnt signaling

doi: 10.1016/j.isci.2023.107012

Figure Lengend Snippet:

Article Snippet: The primary antibodies include anti-active β-Catenin (Millipore, 1:1000), anti-total β-Catenin (Cell signaling, 1:1000), anti-ISL1 (Developmental Studies Hybridoma Bank, 1:1000), anti-Phospho-STAT1 (Tyr701) (Cell signaling, 1:1000), anti-total STAT1 (Cell signaling, 1:1000), anti-GATA4 (Santa Cruz, 1:500), and anti-GAPDH (Ambion,1:5,000).

Techniques: Virus, Functional Assay, Recombinant, Gentle, Plasmid Preparation, Protease Inhibitor, Reverse Transcription, SYBR Green Assay, Knock-Out, Derivative Assay, Software

Journal: Journal of Neuroinflammation

Article Title: The histone deacetylase inhibitor suberoylanilide hydroxamic acid attenuates human astrocyte neurotoxicity induced by interferon-γ

doi: 10.1186/1742-2094-9-113

Figure Lengend Snippet: Effects of SAHA on IFN-γ-induced phosphorylation of STAT1 and STAT3 in human astrocytes. Human astrocytes were incubated with or without SAHA at the concentrations indicated for 1 h. The cells were subsequently stimulated with IFN-γ for 30 minutes. Astrocytes in the control group were incubated with medium only. Cell lysates were separated by 8% SDS-PAGE and immunoblotted for phospho-Tyr 701 -STAT1 (pSTAT1) and total STAT1 (A) or phospho-Tyr 705 -STAT3 (pSTAT3) and total STAT3 (C) . The density ratios of phosphorylated to total protein are shown as mean ± S.E.M. of three independent experiments (B, D) . *Significantly different from IFN-γ stimulation only.

Article Snippet: The protein was transferred to a PVDF membrane at 70 V for 2 h. The membrane was blocked with 5% skim milk plus 3% bovine serum albumin (BSA) in PBS at room temperature (RT) for 1 h. Subsequently, the membrane was incubated with specific rabbit antibodies against phospho-Tyr 701 -STAT1 (1:2,000), total STAT1 (1:1,000), phospho-Tyr 705 -STAT3 (1:2,000) or total STAT3 (1:1,000) at 4°C overnight and then treated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2,000) at RT for 1 h. All antibodies used for immunoblotting were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Incubation, SDS Page