Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage P2Y 6 R activation aggravates psoriatic inflammation through IL-27-mediated Th1 responses

doi: 10.1016/j.apsb.2024.06.008

Figure Lengend Snippet: Macrophage P2Y 6 R mediated Th1 type psoriasis-like inflammation via p-JAK2/p-STAT1/T-bet signaling. (A) Flow diagram. (B, C) FACs and the statistical analysis of IFN γ + CD4 + T-cells ( n = 3). (D) IFN γ concentration after WT or P2Y 6 R −/− mice BMDM supernatant inducing Naïve CD4 + T-cells ( n = 4). (E) The mRNA expression of the Th1-associated cytokines, Ifnγ , Cxcl9 , Cxcl10, and Cxcl11 were detected by RT-qPCR ( n = 3). All mRNA levels were normalized to GAPDH. (F) Linear regression analysis indicated a positive correlation between P2ry6 and Stat1 in GSE181318 and GSE14905. (G) GSEA analysis of GSE50400 showed JNK/STAT signaling pathways were up-regulated in IMQ-induced mice. (H) Top 15 putative transcription factors based on ChEA3. (I, J) The protein expression levels of p-JAK2, p-STAT1, and T-bet in T cells. Normal protein levels were normalized to β -actin, the values for the phosphorylated forms of proteins were normalized to phosphorylation-independent levels of the same protein ( n = 4). (K) CD4 + Naïve T cells supernatant IFN γ concentration after WT or P2Y 6 R −/− mice BMDM supernatant inducing and being treated with or without RO8191 ( n = 4). (L) The proportion of IFN γ + CD4 + T cells of CD4 + Naïve T cells after WT or P2Y 6 R −/− mice BMDM supernatant inducing and being treated with or without RO8191 ( n = 3). (M, N) T-bet protein expression of CD4 + Naïve T cells after WT or P2Y 6 R −/− mice BMDM supernatant inducing and being treated with or without RO8191 ( n = 4). Data are expressed as mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The following antibodies were used: PLC β (bs-6976R, Bioss, Beijing, China), PKC (ab181558, Abcam, Cambridge, UK), p-PKC (EP2730Y, Abcam), p38 (AF6456, Affinity, Jiangsu, China), p-p38 (AF4001, Affinity), ERK (bs-0022R, Bioss), p-ERK (bs-3016R, Bioss), p-JAK2 (A19629, Abclonal, Wuhan, China), JAK2 (AP0531, Abclonal), p-STAT1 (bs1657R, Bioss), STAT1 (10144-2-AP, Proteintech, Wuhan, China), p-JNK (bs1640R, Bioss), JNK (AF6318, Affinity), T-bet (14-5825-82, Abclonal), β -actin (T0022, Affinity).

Techniques: Concentration Assay, Expressing, Quantitative RT-PCR

Journal: Cellular & molecular immunology

Article Title: Avian influenza viruses suppress innate immunity by inducing trans-transcriptional readthrough via SSU72.

doi: 10.1038/s41423-022-00843-8

Figure Lengend Snippet: Fig. 1 TRT enhanced by avian influenza A virus infection represses genes on the complementary DNA strand. A Gene profile of the averaged normalized expression levels of 5,052 genes in A549 cells at 24 h after H1N1/H5N1/H7N9 influenza virus infection or AF treatment. The gene bodies between the TSSs and TTSs were equally sized and scaled to 60 bins, and the gene flanking regions 4 kb upstream of the TSSs and 4 kb downstream of the TTSs were divided into 100-bp windows. B Numbers of TRT genes (FC in the expression level of the TRT region (H5N1/AF) > 5) at different times after H1N1/H5N1/H7N9 infection of A549 cells. C Spearman rank linear correlation coefficient between the upregulated expression levels of the TRT region and the downregulated expression levels of TRT-influenced genes following the trans-TRT and cis-TRT patterns, respectively, at different times after H5N1/H7N9 infection in A549 cells. D Numbers of trans-TRT-influenced genes (FC in the expression level of the trans-TRT gene (H5N1/AF) > 5 and FC in the expression level of the trans-TRT-influenced gene (H1N1/H5N1) > 1.5) at different times after H5N1/H7N9 infection in A549 cells. E Functional pathway enrichment analysis of trans-TRT-influenced genes in H5N1-infected A549 cells (two-tailed P < 0.05, Benjamini–Hochberg adjusted P < 0.05). Detection of F GLS-TRT or G IL23A-TRT in A549 cells by using fluorescence in situ hybridization (FISH). A549 cells were treated with AF/H1N1/H5N1 for 24 h [DAPI nuclear staining (blue) and FISH signals obtained using a Cy3-conjugated DNA probe (red)]. The fluorescence intensity was semiquantitatively assessed using the mean fluorescence intensity (MFI) of each cell. The data are shown as the means ± SEMs. *P < 0.05, **P < 0.01. RNA-seq coverage levels of H the GLS gene, trans-TRT region of GLS, and STAT1 gene, and I the IL23A gene, trans-TRT region of IL23A, and STAT2 gene 12 h after AF/H1N1/H5N1/H7N9 treatment of A549 cells. The gene bodies and intergenic regions, as well as the gene flanking regions 2 kb upstream of the TSSs, were divided into 50-bp windows. Only exon regions are shown in this graph. RNA-seq datasets were established in duplicate

Article Snippet: Primary antibodies specific for the following proteins/peptides were used: SSU72 (1:1000; Cell Signaling Technology, cat. no. 12816) and STAT1 (1:500; Cell Signaling Technology, cat. no. 9172), STAT2 (1:500; Bethyl Laboratories, Inc., Montgomery, Texas, USA; cat. no. A303-512A-M), Flag tag (rabbit, 1:5000; MultiSciences; cat. no. LK-ab002-100), DDDDK tag (mouse, 1:5000; MBL Inc., Ottawa, ON, Canada; cat. no. M185-3 L), β-actin (1:10000; Sigma Aldrich, Saint Louis, MO, USA; cat. no. A5441), and CRISPR/Cas9 (polyclonal; Diagenode Inc., Denville, NJ, USA; cat. no. C15310258).

Techniques: Virus, Infection, Expressing, Functional Assay, Two Tailed Test, In Situ Hybridization, Staining, RNA Sequencing

Journal: Cellular & molecular immunology

Article Title: Avian influenza viruses suppress innate immunity by inducing trans-transcriptional readthrough via SSU72.

doi: 10.1038/s41423-022-00843-8

Figure Lengend Snippet: Fig. 2 TRT inhibition by CRISPR interference enhances STAT1/STAT2 expression and cell viability. RT-qPCR analysis of the A GLS-TRT gRNA and B IL23A-TRT gRNA groups at different times after infection with H5N1 (MOI = 4). RT-qPCR analysis of C STAT1 mRNA expression in the Ctrl gRNA and GLS-TRT gRNA groups and D STAT2 mRNA expression in the Ctrl gRNA and IL23A-TRT gRNA groups at different times after infection with H5N1 (MOI = 4). Western blot analysis of E STAT1 protein expression in the Ctrl gRNA and GLS-TRT gRNA groups and F STAT2 protein expression in the Ctrl gRNA and IL23A-TRT gRNA groups at different times after infection with H5N1 (MOI = 4). β-Actin expression served as the reference control. MTS cell viability assay in the G GLS-TRT gRNA and H IL23A-TRT gRNA groups at 48 h after treatment with AF or infection with H5N1 (MOI = 4). RT-qPCR analysis of viral M2 expression levels in the I GLS-TRT gRNA and J IL23A-TRT gRNA groups at 24 h after infection with H5N1 (MOI = 4). The expression levels in I and J are normalized to the Ctrl gRNA group. Each experiment was repeated at least three times. The data are shown as the means ± SEMs. *P < 0.05, **P < 0.01

Article Snippet: Primary antibodies specific for the following proteins/peptides were used: SSU72 (1:1000; Cell Signaling Technology, cat. no. 12816) and STAT1 (1:500; Cell Signaling Technology, cat. no. 9172), STAT2 (1:500; Bethyl Laboratories, Inc., Montgomery, Texas, USA; cat. no. A303-512A-M), Flag tag (rabbit, 1:5000; MultiSciences; cat. no. LK-ab002-100), DDDDK tag (mouse, 1:5000; MBL Inc., Ottawa, ON, Canada; cat. no. M185-3 L), β-actin (1:10000; Sigma Aldrich, Saint Louis, MO, USA; cat. no. A5441), and CRISPR/Cas9 (polyclonal; Diagenode Inc., Denville, NJ, USA; cat. no. C15310258).

Techniques: Inhibition, CRISPR, Expressing, Quantitative RT-PCR, Infection, Western Blot, Control, Viability Assay

Journal: Cellular & molecular immunology

Article Title: Avian influenza viruses suppress innate immunity by inducing trans-transcriptional readthrough via SSU72.

doi: 10.1038/s41423-022-00843-8

Figure Lengend Snippet: Fig. 4 TRT is reduced and lung injury is ameliorated in SSU72 transgenic mice infected with the lethal H5N1 virus. A Western blot analysis of mouse SSU72 expression in mouse lung tissues at 3 days after treatment with AF/H1N1/H5N1. β-Actin expression served as an internal control. B Numbers of TRT genes (expression of the TRT region upregulated by more than 5 compared with the AF-treated condition) in lung tissues from control (n = 5) and SSU72 transgenic mice (n = 5) at 3 days after intratracheal infection with H5N1 (106 TCID50). The relative mRNA expression ratios of C mouse STAT1 and D STAT2 in lung tissues from control (n = 8) and SSU72 transgenic mice (n = 4) at 3 days after intratracheal infection with H5N1 virus (106 TCID50). Mouse β- actin expression served as the reference control. E Kaplan–Meier survival curves for control (n = 8) and SSU72 transgenic mice (n = 10) after intratracheal infection with H5N1 (106 TCID50). F–H Control and SSU72 transgenic mice were infected with AF or H5N1 (106 TCID50) via intratracheal instillation. F Viral titers in the lungs were assessed 4 days after infection with H5N1 in control (n = 7) and SSU72 transgenic mice (n = 3). G Wet-to-dry weight ratios of the lungs of control (n = 4) and SSU72 transgenic mice (n = 4) at 3 days after infection with H5N1. H Representative images of lung pathology in control and SSU72 transgenic mice at 3 days after H5N1 infection. The lung injury scores (means ± SEMs) and numbers of infiltrating cells per microscopic field (means ± SEMs) are shown in the bar graphs. N = 100 fields for control (n = 15) and SSU72 transgenic (n = 6) mice. Bar = 100 μm. *P < 0.05 and **P < 0.01. Each experiment except for RNA-seq analysis of lungs from mice with or without H5N1 infection was repeated at least three times

Article Snippet: Primary antibodies specific for the following proteins/peptides were used: SSU72 (1:1000; Cell Signaling Technology, cat. no. 12816) and STAT1 (1:500; Cell Signaling Technology, cat. no. 9172), STAT2 (1:500; Bethyl Laboratories, Inc., Montgomery, Texas, USA; cat. no. A303-512A-M), Flag tag (rabbit, 1:5000; MultiSciences; cat. no. LK-ab002-100), DDDDK tag (mouse, 1:5000; MBL Inc., Ottawa, ON, Canada; cat. no. M185-3 L), β-actin (1:10000; Sigma Aldrich, Saint Louis, MO, USA; cat. no. A5441), and CRISPR/Cas9 (polyclonal; Diagenode Inc., Denville, NJ, USA; cat. no. C15310258).

Techniques: Transgenic Assay, Infection, Virus, Western Blot, Expressing, Control, RNA Sequencing

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Use of Inhibitors in the Study of MAP Kinases

doi: 10.1007/978-1-60761-795-2_6

Figure Lengend Snippet: List of antibodies for the phosphorylated and phosphorylation-independent (total) forms of MAP kinases and phosphorylated forms of MAP kinase substrate proteins

Article Snippet: , Stat-1 (S727) , CST (9177).

Techniques: Phospho-proteomics

Journal: Cell reports

Article Title: Janus Kinase 1 Plays a Critical Role in Mammary Cancer Progression

doi: 10.1016/j.celrep.2018.10.063

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal, pY-STAT1 , Origene , Cat# TA309955.

Techniques: Recombinant, Sample Prep, Software

Journal: The Journal of Biological Chemistry

Article Title: A Novel Disrupter of Telomere Silencing 1-like (DOT1L) Interaction Is Required for Signal Transducer and Activator of Transcription 1 (STAT1)-activated Gene Expression

doi: 10.1074/jbc.M111.284190

Figure Lengend Snippet: RNAi-mediated depletion of DOT1L decreases IRF1 gene expression. A, DOT1L Western blot of whole cell extracts collected from 2fTGH cells stably expressing the pTRIPZ shRNAmir non-silencing (shRNA-NS) or shRNAmir-DOT1L (shRNA-DOT1L) vectors with or without IFN-γ. Dynamin served as a loading control. B and C, qRT-PCR to quantitate IRF1 mRNA and heteronuclear RNA (hnRNA) levels in the shRNAmir non-silencing and shRNAmir-DOT1L cell lines treated with IFN-γ for the times indicated or left untreated (Un). IRF1 was normalized to GAPDH, and expression is presented as -fold change relative to the uninduced, shRNAmir-NS condition. Error bars indicate S.E. (n = 3). Student's t test determined significance; **, p ≤ 0.01, *, p ≤ 0.05. D, IRF1 Western blot of whole cell extracts of shRNAmir non-silencing or shRNAmir-DOT1L cells treated with IFN-γ for the indicated times or left untreated. GAPDH served as a loading control. E, H3K79me3 Western blot of acid-extracted histones from the shRNAmir non-silencing and shRNAmir-DOT1L cell lines. Pan H3 served as a loading control. F, STAT1 and phospho-STAT1 Western blots of whole cell extracts collected from 2fTGH cells stably expressing the shRNAmir non-silencing or shRNAmir-DOT1L vectors. The ratio of phospho-STAT1 (pSTAT) to total STAT1 is the same in both cell lines. Western blot bands were quantified with ImageJ.

Article Snippet: Antibodies The antibodies used were: H3K79me3 (Abcam ab2621 for Western blots and Invitrogen 491020 for chromatin immunoprecipitation (ChIP)), Pan H3 CT (Millipore 07-690), RNA polymerase II (Santa Cruz Biotechnology sc-899), IgG (Jackson ImmunoResearch Laboratories), STAT1 (Santa Cruz Biotechnology sc-345X for ChIP, sc-346 for co-immunoprecipitation (co-IP) and Western blots), DOT1L (Bethyl Laboratories A300-953A), FLAG (Sigma F1804), GAPDH (Abcam ab9485), dynamin (Santa Cruz Biotechnology sc7988), phospho-STAT1 (Santa Cruz Biotechnology sc-6402), and anti-rabbit or anti-mouse horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratories).

Techniques: Gene Expression, Western Blot, Stable Transfection, Expressing, shRNA, Control, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: A Novel Disrupter of Telomere Silencing 1-like (DOT1L) Interaction Is Required for Signal Transducer and Activator of Transcription 1 (STAT1)-activated Gene Expression

doi: 10.1074/jbc.M111.284190

Figure Lengend Snippet: RNA polymerase II and STAT1 localization to the IRF1 promoter is reduced in shRNAmir-DOT1L cells. A–H, ChIP of shRNAmir non-silencing (shRNA-NS) or shRNAmir-DOT1L (shRNA-DOT1L) cells treated with IFN-γ for 30 min (right panels) or left untreated (left panels). ChIP was performed with the antibodies indicated, and qPCR, using primers spanning the IRF1 gene locus, quantified the precipitate yield reported as the percentage of input. IgG served as the negative control. p ≤ 0.05 for panels A, B, D, F, and H for solid lines with black diamonds versus dotted lines with black diamonds.

Article Snippet: Antibodies The antibodies used were: H3K79me3 (Abcam ab2621 for Western blots and Invitrogen 491020 for chromatin immunoprecipitation (ChIP)), Pan H3 CT (Millipore 07-690), RNA polymerase II (Santa Cruz Biotechnology sc-899), IgG (Jackson ImmunoResearch Laboratories), STAT1 (Santa Cruz Biotechnology sc-345X for ChIP, sc-346 for co-immunoprecipitation (co-IP) and Western blots), DOT1L (Bethyl Laboratories A300-953A), FLAG (Sigma F1804), GAPDH (Abcam ab9485), dynamin (Santa Cruz Biotechnology sc7988), phospho-STAT1 (Santa Cruz Biotechnology sc-6402), and anti-rabbit or anti-mouse horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratories).

Techniques: shRNA, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: A Novel Disrupter of Telomere Silencing 1-like (DOT1L) Interaction Is Required for Signal Transducer and Activator of Transcription 1 (STAT1)-activated Gene Expression

doi: 10.1074/jbc.M111.284190

Figure Lengend Snippet: STAT1 co-immunoprecipitates with DOT1L. A–C, whole cell extracts prepared from 2fTGH (A), shRNAmir-DOT1L (shRNA-DOT1L) (B), or shRNAmir non-silencing (shRNA-NS) (C) cells were immunoprecipitated (IP) with α-DOT1L, α-STAT1, or IgG (negative control) and then immunoblotted (IB) with the indicated antibodies. A 5% input aliquot of the extracts was included for reference.

Article Snippet: Antibodies The antibodies used were: H3K79me3 (Abcam ab2621 for Western blots and Invitrogen 491020 for chromatin immunoprecipitation (ChIP)), Pan H3 CT (Millipore 07-690), RNA polymerase II (Santa Cruz Biotechnology sc-899), IgG (Jackson ImmunoResearch Laboratories), STAT1 (Santa Cruz Biotechnology sc-345X for ChIP, sc-346 for co-immunoprecipitation (co-IP) and Western blots), DOT1L (Bethyl Laboratories A300-953A), FLAG (Sigma F1804), GAPDH (Abcam ab9485), dynamin (Santa Cruz Biotechnology sc7988), phospho-STAT1 (Santa Cruz Biotechnology sc-6402), and anti-rabbit or anti-mouse horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratories).

Techniques: shRNA, Immunoprecipitation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: A Novel Disrupter of Telomere Silencing 1-like (DOT1L) Interaction Is Required for Signal Transducer and Activator of Transcription 1 (STAT1)-activated Gene Expression

doi: 10.1074/jbc.M111.284190

Figure Lengend Snippet: Mapping of the DOT1L region that interacts with STAT1 in GST pulldown assays. A, graphic depiction of the DOT1L fragments and their relative STAT1 binding affinities. The presence (+) or absence (−) of an interaction is shown, with +, ++, and +++ indicating weak, modest, and strong interactions, respectively. N-term, N-terminal; C-term, C-terminal. B, GST pulldown assays using whole extracts prepared from 2fTGH cells transiently transfected with pcDNA3β FLAG-tagged DOT1L or FLAG-tagged DOT1L fragment vectors and then immunoblotted (IB) with α-FLAG. A 5% input aliquot of the extracts was included for reference.

Article Snippet: Antibodies The antibodies used were: H3K79me3 (Abcam ab2621 for Western blots and Invitrogen 491020 for chromatin immunoprecipitation (ChIP)), Pan H3 CT (Millipore 07-690), RNA polymerase II (Santa Cruz Biotechnology sc-899), IgG (Jackson ImmunoResearch Laboratories), STAT1 (Santa Cruz Biotechnology sc-345X for ChIP, sc-346 for co-immunoprecipitation (co-IP) and Western blots), DOT1L (Bethyl Laboratories A300-953A), FLAG (Sigma F1804), GAPDH (Abcam ab9485), dynamin (Santa Cruz Biotechnology sc7988), phospho-STAT1 (Santa Cruz Biotechnology sc-6402), and anti-rabbit or anti-mouse horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratories).

Techniques: Binding Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: A Novel Disrupter of Telomere Silencing 1-like (DOT1L) Interaction Is Required for Signal Transducer and Activator of Transcription 1 (STAT1)-activated Gene Expression

doi: 10.1074/jbc.M111.284190

Figure Lengend Snippet: Overexpression of the SID of DOT1L represses IRF1 gene expression and alters STAT1 binding. A, qRT-PCR measured IRF1 mRNA expression in 2fTGH, shRNAmir non-silencing (shRNA-NS), and shRNAmir-DOT1L (shRNA-DOT1L) cell lines that were transiently transfected with pcDNA3.0 DOT1L SID, ΔC, ΔN, or empty vector. IFN-γ induction was for 30 min. IRF1 was normalized to GAPDH, and expression is presented as -fold change relative to the uninduced, shRNAmir-NS cell line transfected with empty vector. Error bars indicate S.E. (n = 2). Student's t test determined significance, **, p ≤ 0.01, *, p ≤ 0.05. B, Western blots of acid-extracted histones from the shRNAmir non-silencing and shRNAmir-DOT1L cell lines that were transiently transfected as in panel A. PanH3 served as a loading control. C–H, ChIP, using the indicated antibodies in shRNAmir-DOT1L (right panels) or shRNAmir non-silencing (left panels) cells transiently transfected with pcDNA3.0 DOTL1 SID, ΔC, ΔN, or empty vector and treated with IFN-γ for 30 min. p ≤ 0.05 for black lines with an X or gray squares versus black lines with black squares or white squares in panel C; black lines with gray squares versus others in panel D; black lines with an X or white squares versus black lines with black squares or gray squares in panel E; black lines with white squares versus others in panel F.

Article Snippet: Antibodies The antibodies used were: H3K79me3 (Abcam ab2621 for Western blots and Invitrogen 491020 for chromatin immunoprecipitation (ChIP)), Pan H3 CT (Millipore 07-690), RNA polymerase II (Santa Cruz Biotechnology sc-899), IgG (Jackson ImmunoResearch Laboratories), STAT1 (Santa Cruz Biotechnology sc-345X for ChIP, sc-346 for co-immunoprecipitation (co-IP) and Western blots), DOT1L (Bethyl Laboratories A300-953A), FLAG (Sigma F1804), GAPDH (Abcam ab9485), dynamin (Santa Cruz Biotechnology sc7988), phospho-STAT1 (Santa Cruz Biotechnology sc-6402), and anti-rabbit or anti-mouse horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratories).

Techniques: Over Expression, Gene Expression, Binding Assay, Quantitative RT-PCR, Expressing, shRNA, Transfection, Plasmid Preparation, Western Blot, Control

Journal: ACS Omega

Article Title: Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes

doi: 10.1021/acsomega.4c07915

Figure Lengend Snippet: Molecular docking and dynamic simulations of ROF and JAKs/STATs signaling molecules (A) The binding modes of ROF toward JAK2, JAK3, STAT1, and STAT3. (B–F) The dynamic simulations of JAK2-ROF and JAK3-ROF complexes. The HBond (B), RMSD curves (C), RMSF values (D), Rg values (E), and SASA (F) were analyzed by GROMACS.

Article Snippet: The antibodies for cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, JAK2, p-JAK2, JAK3, p-JAK3, IL-6, STAT1, p-STAT1, STAT3, p-STAT3, and BNIP3 were diluted at a ratio of 1:1000, and β-actin (#BA2305, BOSTER) was diluted at a ratio of 1:5000 in the primary antibody dilution buffer.

Techniques: Binding Assay

Journal: ACS Omega

Article Title: Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes

doi: 10.1021/acsomega.4c07915

Figure Lengend Snippet: Effects of ROF on the JAK2/3-STAT1/3 signaling pathways in Con A-induced hepatitis (A) The phosphorylated levels of JAK2, STAT1, and STAT3 were measured in hepatic tissues using IHC staining ( n = 3; scale bar = 40 μm). (B) The IODs of each protein were detected by using ImageJ. (C) The JAK2/3-STAT1/3 signaling molecules were measured in hepatic tissues with Western blot analysis ( n = 3). The gray value analysis was standardized using the intensity of β-actin and is depicted in bar graphs. The results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs the Con A group.

Article Snippet: The antibodies for cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, JAK2, p-JAK2, JAK3, p-JAK3, IL-6, STAT1, p-STAT1, STAT3, p-STAT3, and BNIP3 were diluted at a ratio of 1:1000, and β-actin (#BA2305, BOSTER) was diluted at a ratio of 1:5000 in the primary antibody dilution buffer.

Techniques: Protein-Protein interactions, Immunohistochemistry, Western Blot

Journal: ACS Omega

Article Title: Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes

doi: 10.1021/acsomega.4c07915

Figure Lengend Snippet: Effects of ROF on Th1/Th17 cells differentiation in vitro (A) The purity of isolated CD4 + T cells was measured by flow cytometry. (B) The viability of CD4 + T cells following 5–20 μM ROF treatments for 24 h was assessed utilizing the CCK-8 assay. (C, D) CD4 + T cells were pre-exposed for 1 h to ROF (20 μM), TOF (1 μM), or ROF (20 μM) + TOF (1 μM), followed by stimulation with Con A for 24 h. The levels of p-JAK2, p-JAK3, p-STAT1, and p-STAT3 were monitored by Western blotting and normalized to each protein’s total concentration in the graphs. (E) The generation of IFN-γ or IL-17 in the cellular supernatants of Th1 or Th17 subtypes were measured using ELISA. (F) The gene expression levels of T-bet or RORγt in differentiated T cells were evaluated by PCR. The values are presented as mean ± SEM ( n = 3). ** P < 0.01, *** P < 0.001 vs the Con A group. # P < 0.05, ## P < 0.01, ### P < 0.001 vs the ROF only group or TOF only group.

Article Snippet: The antibodies for cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, JAK2, p-JAK2, JAK3, p-JAK3, IL-6, STAT1, p-STAT1, STAT3, p-STAT3, and BNIP3 were diluted at a ratio of 1:1000, and β-actin (#BA2305, BOSTER) was diluted at a ratio of 1:5000 in the primary antibody dilution buffer.

Techniques: In Vitro, Isolation, Flow Cytometry, CCK-8 Assay, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: ACS Omega

Article Title: Rhoifolin Attenuates Concanavalin A-Induced Autoimmune Hepatitis in Mice via JAKs/STATs Mediated Immune and Apoptotic Processes

doi: 10.1021/acsomega.4c07915

Figure Lengend Snippet: Effects of ROF on primary hepatocytes apoptosis in vitro (A) Primary hepatocytes were pretreated with ROF (5, 10, 20 μM) for 6 h and treated with or without Con A (10 μg/mL) for 24 h, the cell viability of each group was analyzed by CCK-8 assay. (B) Flow cytometry analysis of apoptosis in primary hepatocytes. (C) Primary hepatocytes were pretreated with ROF (20 μM), TOF (1 μM), or ROF (20 μM) + TOF (1 μM) for 6 h, and subsequently triggered with Con A (10 μg/mL) for 24 h. The IL-6, p-JAK2, p-STAT1, p-STAT3, BNIP3, and Bax expression levels were evaluated by using Western blotting. (D) The quantification and standardization of the protein expression were performed using ImageJ. The results are presented as mean ± SEM of three independent experiments. ** P < 0.01, *** P < 0.001 vs the Con A group. # P < 0.05, ## P < 0.01 vs the ROF only group or TOF only group.

Article Snippet: The antibodies for cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, JAK2, p-JAK2, JAK3, p-JAK3, IL-6, STAT1, p-STAT1, STAT3, p-STAT3, and BNIP3 were diluted at a ratio of 1:1000, and β-actin (#BA2305, BOSTER) was diluted at a ratio of 1:5000 in the primary antibody dilution buffer.

Techniques: In Vitro, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal:

Article Title: Signaling of short- and long-term regulation of intestinal epithelial type 1 Na + /H + exchanger by interferon- γ

doi: 10.1038/sj.bjp.0706167

Figure Lengend Snippet: Abundance of total STAT1, phospho-STAT1 and phospho-p38 MAPK in Caco-2 cells treated with vehicle and IFN-γ (1000 U ml−1) or anisomycin (100 nM) for 1, 3 and 24 h.

Article Snippet: Total and phosphorylated STAT1 protein levels in Caco-2 cells exposed to IFN- γ (1000 U ml −1 ) and anisomycin (100 n M ) for 1, 3 and 24 h were analyzed using the PhosphoPlus® Stat1 (Tyr701) antibody kit (Cell Signaling TechnologyTM) according to the manufacturer's protocol.

Techniques:

Journal:

Article Title: Signaling of short- and long-term regulation of intestinal epithelial type 1 Na + /H + exchanger by interferon- γ

doi: 10.1038/sj.bjp.0706167

Figure Lengend Snippet: Effect of EGCG (20 μM), SB 203580 (10 μM) and PKC downregulation (PKC dr; overnight treatment with 100 nM PDBu) on the abundance of STAT1 and phospho-STAT1 in Caco-2 cells treated for 24 h in the absence (vehicle) and presence of IFN-γ (1000 U ml−1).

Article Snippet: Total and phosphorylated STAT1 protein levels in Caco-2 cells exposed to IFN- γ (1000 U ml −1 ) and anisomycin (100 n M ) for 1, 3 and 24 h were analyzed using the PhosphoPlus® Stat1 (Tyr701) antibody kit (Cell Signaling TechnologyTM) according to the manufacturer's protocol.

Techniques: