Journal: International Journal of Molecular Sciences

Article Title: Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition

doi: 10.3390/ijms23073980

Figure Lengend Snippet: Canonical or non-canonical STAT1 pathway protein expression. Cells were activated with IFN-γ or IFN-γ+TNF-α and treated with 2 mM butyrate (green bars) for 1 h, 4 h or 16 h. IRF9 ( A , E , F ), phosphorylated JAK2 ( B , G – I ), phosphorylated STAT1 ( C , J – L ) and phosphorylated NFkB p65 ( D , M – O ) protein expression was determined. Representable blots of proteins of interest and β-actin control are shown in A-D. Data are represented as mean ± SEM ( n = 3). Significant differences are shown as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, medium control as compared to 2 mM butyrate control or to IFN-γ or IFN-γ+TNF-α-activated intestinal epithelial cells in absence of butyrate. Activated cells were also compared to activated cells treated with butyrate.

Article Snippet: As primary antibodies phosphorylated JAK2 (1:200, Thermo Fisher Scientific), phosphorylated STAT1 (1:200, R&D systems), phosphorylated NF-kappaB p65 (1:1000, Cell signaling, Danvers, MA, USA), IRF9 (1:1000, Cell signaling) and β-actin (1:1000, Cell signaling) were used.

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition

doi: 10.3390/ijms23073980

Figure Lengend Snippet: The effect of butyrate and histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) on mRNA expression of genes related to the STAT1 signaling cascade. Intestinal epithelial cells were activated with IFN-γ or IFN-γ+TNF-α and treated with 2 mM butyrate (green bars) or 10 μM TSA (pink bars) for 4 h. mRNA expression was measured in CXCL10 ( A , B ), IRF9 ( C , D ) JAK2 ( E , F ) and SOCS1 ( G , H ). Data are represented as mean ± SEM ( n = 4). Significant differences are shown as ** p < 0.01, *** p < 0.001, **** p < 0.001 Control compared to butyrate, TSA, IFN-γ ( A , C , E , G ) or IFNγ+TNFα ( B , D , F , H ) activated cells and activated cells compared to butyrate or TSA treated activated cells.

Article Snippet: As primary antibodies phosphorylated JAK2 (1:200, Thermo Fisher Scientific), phosphorylated STAT1 (1:200, R&D systems), phosphorylated NF-kappaB p65 (1:1000, Cell signaling, Danvers, MA, USA), IRF9 (1:1000, Cell signaling) and β-actin (1:1000, Cell signaling) were used.

Techniques: Histone Deacetylase Assay, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition

doi: 10.3390/ijms23073980

Figure Lengend Snippet: The effect of histone deacetylase inhibitor Trichostatin A (TSA) on downstream proteins of the STAT1 signaling cascade. Protein expression in IFN-γ or IFN-γ+TNF-α-activated intestinal epithelial cells (IECs) after 4 h incubation with 10 μM TSA (pink bars). Proteins measured were IRF9 ( A , E ), phosphorylated JAK2 ( B , F ), phosphorylated STAT1 ( C , G ), phosphorylated NFκB p65 ( D , H ). Data are represented as mean ± SEM ( n = 3). Significant differences are shown as * p < 0.05, ** p < 0.01, *** p < 0.001, control compared to IFN-γ-activated IECs, IFN-γ+TNF-α-activated IECs or 10 μM TSA control. Activated cells were compared to activated cells treated with TSA.

Article Snippet: As primary antibodies phosphorylated JAK2 (1:200, Thermo Fisher Scientific), phosphorylated STAT1 (1:200, R&D systems), phosphorylated NF-kappaB p65 (1:1000, Cell signaling, Danvers, MA, USA), IRF9 (1:1000, Cell signaling) and β-actin (1:1000, Cell signaling) were used.

Techniques: Histone Deacetylase Assay, Expressing, Incubation, Control

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 1 DHX9 expression significantly increased in oxLDL or interferon-γ-treated macrophages and peripheral blood mononuclear cells from patients with coronary artery disease. a Single-cell RNA analysis of DHX9 mRNA expressions in mononuclear phagocytes (MNPs) a public MNP single-cell RNA compendium. b Western blot analysis of DHX9 expressions PBMCs from health volunteers stimulated with 50 ng/ml of recombi- nant human GM-CSF for different hours. c Western blot analysis of DHX9 expressions in the THP-1-derived macrophages stimulated with oxLDL for 24 h. d Western blot analysis of DHX9 expressions in the THP-1-derived macrophages stimulated with 40 μg/ml oxLDL for different hours. e Western blot analysis of DHX9, STAT1, p-STAT1 expressions in the THP-1-derived macrophages stimulated with 20 ng/mL IFN-γ for different hours. GAPDH was used as a loading control. f Immunofluorescence analysis of DHX9 cellular distribution in macrophages treated with or without 40 μg/mL oxLDL for 24 h. Scale bars, 10 μm. g Western blot analysis of DHX9 expressions in PBMCs isolated from health volunteers (HV) or patients with (CAD). GAPDH was used as a loading control.

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Expressing, Western Blot, Derivative Assay, Control, Immunofluorescence, Isolation

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 2 Knockdown of DHX9 inhibits lipid uptake and pro-inflammatory factor expressions of macrophages, and ameliorates TNF-α-mediated monocyte adhesion capacity. a qPCR detection of IL-6 and TNF mRNAs in THP-1-derived macrophages transfected with control siRNA (si-NC) or siRNA against DHX9 (si-DHX9) for 48 h. b Flow cytometry analysis of cell cycle of THP-1-derived macrophages transfected with control siRNA (si-NC) or siRNA against DHX9 (si-DHX9) for 48 h. c Flow cytometry analysis of cell apoptosis of THP-1-derived macrophages transfected with control siRNA (si-NC) or siRNA against DHX9 (si-DHX9) for 48 h. d Immunofluorescence analysis of Dil-oxLDL (red) uptake of THP-1-derived macrophages transfected si-NC or si-DHX9 for 48 h. Scale bars, 5 μm. e qPCR detection of IL-1β, IL-6 and TNF-α mRNAs in THP-1-derived mac- rophages transfected with si-NC or si-DHX9 for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. Data are represented as means ± SD (n = 3; * P < 0.05 vs. si-nc + oxLDL group). f and g Correlation analysis of DXH9 and IL-6 or TNFα gene expressions in monocytes. h Representative images of the attachment of THP-1 cells transfected si-NC or si-DHX9 to HUVECs. Scale bars, 160 μm. Data are represented as means ± SD. n = 3; Statistical differences were calculated using unpaired two-tailed Student’s t test. *P < 0.05.

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Knockdown, Derivative Assay, Transfection, Control, Flow Cytometry, Immunofluorescence, Incubation, Two Tailed Test

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 3 oxLDL stimulation promotes DHX9 interaction with p65 in macrophages. a Western blotting analysis of p38, JNK, ERK signaling in mac- rophages transfected with si-DHX9 or si-nc for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. b Western blotting analysis of NF-κB signaling in macrophages transfected with si-DHX9 or si-nc for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. c Co-IP detection of the interaction between DHX9 and p65 in macrophages treated with or without 40 μg/mL oxLDL for 24 h by using DHX9 antibody. d Immunofluores- cence analysis of DHX9 and P65 expression in macrophages treated with or without 40 μg/mL oxLDL. Scale bars, 10 μm. e Co-IP detection of the interaction between DHX9 and p65 in the nuclear fractions of macrophages by using DHX9 antibody. f P65 dimer formation was detected using naïve PAGE when macrophages were transfected with or without FLAG-DHX9 and treated with or without oxLDL (-, 0 μg/mL oxLDL; +, 40 μg/ mL oxLDL; ++, 80 μg/mL oxLDL).

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Western Blot, Transfection, Incubation, Co-Immunoprecipitation Assay, Expressing

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 4 oxLDL stimulation promotes the transcriptional activity of DHX9-p65-RNA Polymerase II complex. a ChIP analysis of the binding of DHX9 to IL-6 promoter in macrophages treated with oxLDL (-, 0 μg/mL oxLDL; +, 40 μg/mL oxLDL; ++, 80 μg/mL oxLDL). IgG was used as the control of anti-DHX9. Data are represented as means ± SD (n = 3; *P < 0.05). b and c ChIP-re-ChIP analysis of the binding of DHX9-p65 com- plex to IL-6 promoter in macrophages treated with oxLDL. d ChIP analysis of the binding of RNA Polymerase II to IL-6 promoter in macrophages transfected with si-DHX9 or si-nc for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. e ChIP analysis of the binding of p65 to IL-6 promoter in macrophages transfected with si-DHX9 or si-nc for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. f and g ChIP-re-ChIP analysis of the binding of RNA Polymerase II-p65 complex to IL-6 promoter in macrophages transfected with si-DHX9 or si-nc for 48 h and later incubated with 40 μg/mL oxLDL for 24 h. Data are represented as means ± SD (n = 3; *P < 0.05).

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Activity Assay, Binding Assay, Control, Transfection, Incubation

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 5 Knockdown of DHX9 alleviates AS progression in vivo. a Western blot analysis of DHX9 protein expression in the arterial tissues of AAV- sh-DHX9 group mice and AAV- sh-NC group mice. b Representative images and quantification of the aorta en face lesion stained with oil red O (n = 6 for each group). Data are represented as means ± SD (n = 6; *P < 0.05). c Representative images and quantification of the aortic root lesion area stained with oil red O (n = 6 for each group). Data are represented as means ± SD (n = 6; *P < 0.05). d Immunofluorescence analysis of F4/80 and p-p65 in the plaques of arteries of AAV- sh-DHX9 group mice and AAV- sh-NC group mice. Scale bars, 20 μm. e qPCR detection of IL-6 and TNF-α mRNA expressions in the plaques of arteries of AAV- sh-DHX9 group mice and AAV- sh-NC group mice. g and f ELISA detection of IL-6 and TNF-α expressions in the plasma of mice. Data are represented as means ± SD (n = 3; *P < 0.05).

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Knockdown, In Vivo, Western Blot, Expressing, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 6 Knockdown of DHX9 inhibits p65 activation, inflammatory factor expressions, and the transcriptional activity of p65-RNA Polymerase II complex in PBMCs from CAD patients. a Western blot analysis of DHX-9 and p-p65 protein expressions in the PBMCs from CAD patients trans- fected with si-DHX9 or si-NC. b qPCR detection of IL-6 and TNF-α mRNA expressions in the PBMCs from CAD patients transfected with si- DHX9 or si-NC. c ChIP analysis of the binding of DHX9 to IL-6 promoter in PBMCs. d ChIP analysis of the binding of RNA Polymerase II to IL-6 promoter in the PBMCs from CAD patients transfected with si-DHX9 or si-NC. e ChIP analysis of the binding of p65 to IL-6 promoter in the PBMCs from CAD patients transfected with si-DHX9 or si-NC. Data are represented as means ± SD (n = 3; *P < 0.05).

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Knockdown, Activation Assay, Activity Assay, Western Blot, Transfection, Binding Assay

Journal: Inflammation

Article Title: DHX9 Strengthens Atherosclerosis Progression By Promoting Inflammation in Macrophages.

doi: 10.1007/s10753-023-01836-z

Figure Lengend Snippet: Fig. 7 DHX9 promotes ox-LDL-induced inflammation in macrophages via interacting with p65. DHX9 interacts with p65 in ox-LDL-stimulated macrophages to enhance the transcriptional activity of DHX9-p65-RNA Polymerase II complex to produce inflammatory factors.

Article Snippet: 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), PhosphoP38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), PhosphoJNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech).

Techniques: Activity Assay

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: 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: 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: 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: