Journal: Scientific Reports

Article Title: Comparison of anti-inflammatory and anti-angiogenic effects of JAK inhibitors in IL-6 and TNFα-stimulated fibroblast-like synoviocytes derived from patients with RA

doi: 10.1038/s41598-025-94894-2

Figure Lengend Snippet: ( a ) Western blotting was performed to analyze STAT1, pSTAT1, STAT3, pSTAT3, and β-actin. ( b , c ) Comparison of the ratios of pSTAT1 to STAT1 and pSTAT3 to STAT1. Protein expression was determined by semi-quantitative analysis of digitally captured images. All JAK inhibitors significantly suppressed pSTAT1 and pSTAT3 levels compared to those in the control. Compared to TOF, BAR, PEF, UPA, and FIL suppressed pSTAT1 and pSTAT3 levels. STAT, signal transducer and activator of transcription; JAK, Janus kinase; TOF, tofacitinib; BAR, baricitinib; PEF, peficitinib; UPA, upadacitinib; FIL, filgotinib.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBST at 25 °C for 30 min, incubated with antibodies against anti-STAT1, anti-phosphorylated STAT1 (pSTAT1), anti-STAT3, and anti-pSTAT3 (Cell Signaling Technology, Danvers, MA, US) at 4 °C for 12 h, and further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody at 25 °C for 1 h. The proteins were subsequently visualized using ECL Plus reagent (GE Healthcare Life Sciences, Little Chalfont, UK) on a chemiluminescence analyzer (LAS-3000 mini; Fujifilm, Tokyo, Japan).

Techniques: Western Blot, Comparison, Expressing, Control

Journal: Advanced Science

Article Title: Gene Therapy for Inflammatory Cascade in Intrauterine Injury with Engineered Extracellular Vesicles Hybrid Snail Mucus‐enhanced Adhesive Hydrogels

doi: 10.1002/advs.202410769

Figure Lengend Snippet: Schematic representation of siRNA@FA‐EVs/GS hydrogel inhibited M1 macrophage polarization and pro‐inflammatory cytokines production through the inhibition of STAT1 phosphorylation, which reduced the transformation of endometrial stromal cells into myofibroblasts and collagen deposition, and inhibited the formation of intrauterine adhesion.

Article Snippet: The anti‐phosphorylated STAT1 (pSTAT1) and anti‐CD86 were bought from HUABIO (Hangzhou, China).

Techniques: Inhibition, Phospho-proteomics, Transformation Assay

Journal: Advanced Science

Article Title: Gene Therapy for Inflammatory Cascade in Intrauterine Injury with Engineered Extracellular Vesicles Hybrid Snail Mucus‐enhanced Adhesive Hydrogels

doi: 10.1002/advs.202410769

Figure Lengend Snippet: A, B) Heatmap analysis of proteomics and transcriptomic dataset. C) Volcano plot exhibited DEGs and DEPs in the transcriptomic dataset and proteomics dataset. D) Gene set enrichment analysis (GSEA) of genes altered GO and KEGG pathway analysis. E) Venn diagram analysis of the intersection of transcriptomic data and proteomics data. F) Immunofluorescence co‐localization of pSTAT1 in M1 (CD86‐labeled) and M2 (Arg1‐labeled) macrophages in IUA. G) STAT1 binding sites in the promoter region of TNF‐α and IL‐1β. H) The concentration of TNF‐α and IL‐1β in the disease course (0, 1, 3, 7, 14, 28 days after surgery), Data are presented as mean ± SD, n = 3.

Article Snippet: The anti‐phosphorylated STAT1 (pSTAT1) and anti‐CD86 were bought from HUABIO (Hangzhou, China).

Techniques: Immunofluorescence, Labeling, Binding Assay, Concentration Assay

Journal: Advanced Science

Article Title: Gene Therapy for Inflammatory Cascade in Intrauterine Injury with Engineered Extracellular Vesicles Hybrid Snail Mucus‐enhanced Adhesive Hydrogels

doi: 10.1002/advs.202410769

Figure Lengend Snippet: A,B) The phosphorylation level of STAT1 assessed by fluorescence microscopy and flow cytometric assays. C) The fluorescence intensity ratio of pSTAT1 in the nucleus of macrophages. D) The percentage of pSTAT1‐positive macrophages was detected by flow cytometry. E) Schematic representation of siRNA@FA‐EVs/GS inhibited M1 macrophage polarization by inhibiting STAT1 phosphorylation. Data are presented as mean ± SD. * p < 0.05 compared to the control group, ** p < 0.05 compared to the LPS+GS group, *** p < 0.05 compared to the LPS+siRNA@EVs/GS group, n = 6.

Article Snippet: The anti‐phosphorylated STAT1 (pSTAT1) and anti‐CD86 were bought from HUABIO (Hangzhou, China).

Techniques: Phospho-proteomics, Fluorescence, Microscopy, Flow Cytometry, Control

Journal: Advanced Science

Article Title: Gene Therapy for Inflammatory Cascade in Intrauterine Injury with Engineered Extracellular Vesicles Hybrid Snail Mucus‐enhanced Adhesive Hydrogels

doi: 10.1002/advs.202410769

Figure Lengend Snippet: A,B) Heatmap and volcano plots of DEGs. C) GSEA analysis was applied to perform function annotation. D) The normalized count values of the STAT1, IL‐1β, and TNF‐α E) The protein bands of pSTAT1 and CD86. F) The immunofluorescence images of pSTAT1/CD86 in IUA tissues. G) The percentage of pSTAT1/CD86 positive cells evaluated by immunofluorescence staining in each group. H, I) The protein expression of pSTAT1 and CD86 was detected. Data are presented as mean ± SD. * p < 0.05 compared with IUA+GS; ** p < 0.05 compared with IUA+siRNA@EVs/GS group, ns, no significance between two groups, n = 6.

Article Snippet: The anti‐phosphorylated STAT1 (pSTAT1) and anti‐CD86 were bought from HUABIO (Hangzhou, China).

Techniques: Immunofluorescence, Staining, Expressing

Journal: bioRxiv

Article Title: The Unexpected Match: STAT3-NORAD Interaction, a Novel Link in Antiviral Defense

doi: 10.1101/2023.06.29.546999

Figure Lengend Snippet: (A) Single-molecule Fluorescence In Situ Hybridization (smFISH) of the long non-coding RNA (lncRNA) NORAD in hESCs. The FISH data demonstrates that NORAD is predominantly located in the cytoplasm with a 2.2-fold higher concentration than in the nucleus. This suggests a significant role for NORAD in cytoplasmic processes. (B) NORAD knockdown efficiency in hESCs and HFFs, detected by rt-qPCR. (C) Volcano plot illustrating differentially expressed ISGs between hESCs and HFFs. The plot reveals a greater expression of ISGs in HFFs as compared to hESCs, indicating a differential baseline of the innate immune responses between these two cell types. (D, E) ChIP Enrichment Analysis (ChEA3) of differentially expressed genes upon knockdown of NORAD in (D) hESCs and (E) HFFs. Bar plots and network visualizations are provided. As shown, in both hESC and HFFs STAT1, STAT2, and STAT3 are at the top of the list of transcription factors that are predicted to regulate these differentially expressed genes. Results highlight the potential broad influence of NORAD on STAT-mediated signaling pathways.

Article Snippet: Western blot analysis was performed using antibodies against STAT1, STAT3, phosphorylated STAT1 (pSTAT1) at residue 701, and phosphorylated STAT3 (pSTAT3) at residues 705 and 727 (Cell Signaling Cats:#14994, #12640, #7649,#9145,#9134 respectively).

Techniques: Fluorescence, In Situ Hybridization, Concentration Assay, Knockdown, Quantitative RT-PCR, Expressing, Protein-Protein interactions

Journal: bioRxiv

Article Title: The Unexpected Match: STAT3-NORAD Interaction, a Novel Link in Antiviral Defense

doi: 10.1101/2023.06.29.546999

Figure Lengend Snippet: (A and B) Left: Volcano plot of RNA-seq data from NORAD KD versus control siRNA. Differentially expressed (Padj<0.05, -2>FC>2) ISGs are marked in red. Right: Gene ontology enrichment analyses of the DEGs upon NORAD KD. (A) hESCs 72 hours post siRNA transfection (n=4) (B) HFFs 24 hours post transfection (n=4). (C) heatmap of FCs of ISGs 8, 16, 24 hours post transfection in HFFs, shown are only differentially expressed ISGs in at least one time point (D) Scatterplots demonstrating the correlations between FCs calculated for all expressed ISGs in IAV relative to mock-infected cells and the FCs calculated for ISG expression in NORAD KD in this study. Up: Comparison between IAV infection in hIPSCs to siNORAD treatment in hESCs. Down: Comparison between IAV infection to NORAD KD in HFFs. (E-F) RT-qPCR analysis of ISGs expression in double KD experiments in HFFs of STAT3+NORAD (E) and STAT1+NORAD (F) . siNORAD (red), siSTAT (light blue) and siSTAT + siNORAD (yellow) relative to siCTRL. (G) Western blot analysis of STAT3 and STAT1 protein expression in HFFs 24 hours post siRNA transfection.

Article Snippet: Western blot analysis was performed using antibodies against STAT1, STAT3, phosphorylated STAT1 (pSTAT1) at residue 701, and phosphorylated STAT3 (pSTAT3) at residues 705 and 727 (Cell Signaling Cats:#14994, #12640, #7649,#9145,#9134 respectively).

Techniques: RNA Sequencing, Control, Transfection, Infection, Expressing, Comparison, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: The Unexpected Match: STAT3-NORAD Interaction, a Novel Link in Antiviral Defense

doi: 10.1101/2023.06.29.546999

Figure Lengend Snippet: (A and B) An immunofluorescence visualization - upon NORAD knockdown compared to siCTRL samples in HFFs (16h post siRNA transfection), (A) STAT3 (B) STAT1 (In blue: nuclear staining by DAPI). (C) Box plots of the ratio between the nucleus and cytoplasm IF signal (55 and 61 cells for STAT3 siCTRL and siNORAD, respectively and 71 and 64 cells for STAT1 siCTRL and siNORAD, respectively) showing significant differences between the different conditions (STAT3 p-value = 8.1 e-10, STAT1 p-value = 9.7e-9, Mann Whitney Wilcoxon test). (D) Western blot analysis of Co-IP experiment. HFF lysates from siNORAD and siCTRL samples immunoprecipitated by anti-STAT3. Protein-protein interactions were immunodetected by anti-Importin-β1, anti-STAT3 and anti-GAPDH. (E) Binding enrichment of STAT3 (top) and STAT1 (bottom) to the promoters of six selected ISGs in siCTRL and siNORAD in HFFs. DNA binding levels were determined by ChIP-qPCR (n=2). (F) Illustration of the proposed model for the role of NORAD-STAT3-interaction in regulating ISG transcription.

Article Snippet: Western blot analysis was performed using antibodies against STAT1, STAT3, phosphorylated STAT1 (pSTAT1) at residue 701, and phosphorylated STAT3 (pSTAT3) at residues 705 and 727 (Cell Signaling Cats:#14994, #12640, #7649,#9145,#9134 respectively).

Techniques: Immunofluorescence, Knockdown, Transfection, Staining, MANN-WHITNEY, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Protein-Protein interactions, Binding Assay, ChIP-qPCR

Journal: bioRxiv

Article Title: The Unexpected Match: STAT3-NORAD Interaction, a Novel Link in Antiviral Defense

doi: 10.1101/2023.06.29.546999

Figure Lengend Snippet: (A , B) An immunofluorescence visualization - upon NORAD knockdown compared to siCTRL samples in hESCs (72h post siRNA transfection), (A) STAT3 (B) STAT1 (In blue: nuclear staining by DAPI). (C) Box plots of the ratio between the nucleus and cytoplasm IF signal (282 and 302 cells for STAT3 siCTRL and siNORAD, respectively and 59 and 67 cells for STAT1 siCTRL and siNORAD, respectively) showing significant differences between the different conditions (STAT3 p<3.76e-23, STAT1 p<0.0157, Mann Whitney Wilcoxon test)

Article Snippet: Western blot analysis was performed using antibodies against STAT1, STAT3, phosphorylated STAT1 (pSTAT1) at residue 701, and phosphorylated STAT3 (pSTAT3) at residues 705 and 727 (Cell Signaling Cats:#14994, #12640, #7649,#9145,#9134 respectively).

Techniques: Immunofluorescence, Knockdown, Transfection, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: The Unexpected Match: STAT3-NORAD Interaction, a Novel Link in Antiviral Defense

doi: 10.1101/2023.06.29.546999

Figure Lengend Snippet: (A) qPCR analysis of Norad knockdown in MEF cells. Demonstrating that KD of Norad does not change the RNA levels of Stat3, Stat1, and Isg15 (B) A volcano plot of RNA-seq data from Norad KD versus control siRNA in MEF cells 24 hours post-transfection. Differentially expressed (Padj<0.05, -2>FC>2) ISGs are marked in red. (C) Western blot analysis of Stat3 CLIP-qPCR in MEF cells. 5% of the IP taken for the analysis (D) 2D representation of the sequences of the STAT3 binding sites on NORAD from different species. Sequences were folded using the LocARNA Alignment & Folding prediction tool. The structure with the highest conservation was predicted for sequences from ALU-containing primates, significantly higher than for sequences extracted from non-ALU primates, Rodents, and other Mammals.

Article Snippet: Western blot analysis was performed using antibodies against STAT1, STAT3, phosphorylated STAT1 (pSTAT1) at residue 701, and phosphorylated STAT3 (pSTAT3) at residues 705 and 727 (Cell Signaling Cats:#14994, #12640, #7649,#9145,#9134 respectively).

Techniques: Knockdown, RNA Sequencing, Control, Transfection, Western Blot, Binding Assay

Journal: Frontiers in Immunology

Article Title: Identification of a Novel Mutation in TNFAIP3 in a Family With Poly-Autoimmunity

doi: 10.3389/fimmu.2022.804401

Figure Lengend Snippet: Interferon gamma pathway is upregulated in HA20 patients. (A) CXCL9 and CXCL10 levels were measured in plasma samples collected from patients (Pt1-4) during different hospitalizations, from the father and from healthy donors (HD; n=15) by ELISA. Red bars indicate sample median. Statistical analyses were performed with Mann-Whitney test comparing each patient with HDs. **p<0,01; ***p<0,001. (B) Unstimulated PBMCs from patients (Pt1-4) and two healthy donors (HDs) were stained for total STAT1 levels. Results are reported as STAT1 mean fluorescence intensity (MFI) in monocytes (CD14+ cells). (C) PBMCs from patients (Pt1-4) and two healthy donors (HDs) were stimulated for 10 minutes with 10 ng/ml of IFNγ and phosphorylated STAT1 (pSTAT1) levels were detected by flow cytometry. Results were reported as % of pSTAT1 positive monocytes (CD14+ cells). (D) PBMCs from patients (Pt1-4) and one healthy donor (HD) were left unstimulated (US, white columns) or stimulated for 2 hours with 10 ng/ml of IFNγ (IFNγ, black columns) and CXCL9 and CXCL10 mRNA levels were analyzed by qPCR. Results were obtained after normalization with the housekeeping gene HPRT1 and were expressed as arbitrary units (AU). Similar results were obtained in two independent experiments.

Article Snippet: After permeabilization with Perm Buffer II (BD PhosFlow) 20 min at 4°C, samples were stained with antibodies against phosphorylated Tyrosine (701) STAT1 (pSTAT1) and total STAT1 (all from Becton Dickinson) for 20 min at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Staining, Fluorescence, Flow Cytometry