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: 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: Phospho-proteomics, Incubation, Control, SDS Page

Journal: International Journal of Molecular Sciences

Article Title: CD26 Deficiency Controls Macrophage Polarization Markers and Signal Transducers during Colitis Development and Resolution

doi: 10.3390/ijms23105506

Figure Lengend Snippet: The influence of CD26 deficiency on mRNA and protein expression of STAT transcription factors in colonic tissue during dextran sulfate sodium (DSS)-induced colitis ( a ) Relative quantification of STAT1, STAT3, and STAT6 genes expression in colonic tissue of C57BL/6 and CD26 −/− mice with DSS-induced colitis was determined by quantitative real-time (RT)-PCR on days 3, 7, and 15 days after colitis induction. The RT-PCR analysis was performed in duplicate. ( b ) Protein expression of STAT1, STAT3, and STAT6 and their phosphorylated forms in colonic tissue of C57BL/6 and CD26 −/− mice with DSS-induced colitis was performed by Western blot analysis on days 3, 7, and 15 days after colitis induction. ( c ) Relative quantification of the expression ratio of proteins phospho-STAT1 and STAT1, phospho-STAT3 and STAT3, and phospho-STAT6 and STAT6. Data are expressed as mean ± SD. n = 6 mice/each group. a, b, and c denote p < 0.05, p < 0.01, and p < 0.001 compared with the control group, respectively. Comparisons between related DSS-treated C57BL/6 and CD26 −/− groups are highlighted by horizontal lines above the bars.

Article Snippet: After blocking in 5% nonfat dry milk, the membranes were incubated overnight at 4 °C with primary antibodies against STAT1 (1:1000, #14994, Cell Signaling Technology, Danvers, MA, USA), phospho-STAT1 (Tyr701) (1:1000, #7649, Cell Signaling Technology, Danvers, MA, USA), STAT3 (1:1000, #12640, Cell Signaling Technology), phospho-STAT3 (Tyr705) (1:1000, ab76315, Abcam, Cambridge, UK), STAT6 (1:1000, #5397, Cell Signaling Technology, Danvers, MA, USA), phospho-STAT6 (Tyr641) (1:1000, #56554, Cell Signaling Technology, Danvers, MA, USA) and β-actin (1:10000, ab8226, Abcam, Cambridge, UK).

Techniques: Expressing, Quantitative Proteomics, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: CD26 Deficiency Controls Macrophage Polarization Markers and Signal Transducers during Colitis Development and Resolution

doi: 10.3390/ijms23105506

Figure Lengend Snippet: Forward and reverse primer sequences used in quantitative real-time (RT)-PCR analysis of the expression of genes of interest.

Article Snippet: After blocking in 5% nonfat dry milk, the membranes were incubated overnight at 4 °C with primary antibodies against STAT1 (1:1000, #14994, Cell Signaling Technology, Danvers, MA, USA), phospho-STAT1 (Tyr701) (1:1000, #7649, Cell Signaling Technology, Danvers, MA, USA), STAT3 (1:1000, #12640, Cell Signaling Technology), phospho-STAT3 (Tyr705) (1:1000, ab76315, Abcam, Cambridge, UK), STAT6 (1:1000, #5397, Cell Signaling Technology, Danvers, MA, USA), phospho-STAT6 (Tyr641) (1:1000, #56554, Cell Signaling Technology, Danvers, MA, USA) and β-actin (1:10000, ab8226, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Journal of Virology

Article Title: Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation

doi: 10.1128/JVI.01136-17

Figure Lengend Snippet: Inhibition of nuclear accumulation of STAT1 and -2 in the presence of NiV-N protein. (A) Cos7 or HeLa cells were transfected with 0.6 μg of the pCAGGS-NiV-N or -NiV-N-HA expression plasmid and were treated with IFN-α or -γ for 30 min. The cells were stained using anti-STAT1, -pSTAT1, -STAT2, and -pSTAT2 antibodies with an anti-N polyclonal antibody (or anti-HA mouse antibody) and Hoechst 33342 dye. The images shown are all z-stack data. Arrowheads indicate the cytoplasmic STATs in the NiV-N-expressing cells. The bar graphs show the statistical evaluation of STAT nuclear accumulation. We compared the scores for STAT distribution between NiV-N-expressing and non-NiV-N-expressing cells. The scores were calculated by counting approximately 60 cells from at least five randomly selected fields. Filled bars indicate the percentage of nuclear localization, while white bars indicate the percentage of whole-cell distribution. Error bars indicate standard deviations. The experiments were repeated three times independently. **, P < 0.01; ***, P < 0.001.

Article Snippet: The following antibodies were purchased: rabbit antibodies against STAT1 p84/p91 (E-23 [sc-346]; Santa Cruz), phospho-STAT1 (Tyr701) (9171; Cell Signaling Technology), STAT2 (C-20 [sc-476]; Santa Cruz), p-Stat2 (Tyr690) (sc-21689-R; Santa Cruz), STAT3 (H-190 [sc-7179]; Santa Cruz), histone H3 (D1H2) (4499P; Cell Signaling Technology), tubulin (H300 [sc-5546]; Santa Cruz), PKR (D-20 [sc-708]; Santa Cruz), HA tag (631207; Clontech), and c-Myc tag (C3956; Sigma-Aldrich); a rabbit IgG isotype control antibody (ab37415-5; Abcam); and mouse antibodies against STAT1α p91 (C-111 [sc-417]; Santa Cruz), STAT2 (A-7 [sc-1668] and A-9 [sc-166201]; Santa Cruz), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (MAB374; Millipore), HA tag (H9654; Sigma-Aldrich), and c-Myc (631206; Clontech).

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Staining

Journal: Journal of Virology

Article Title: Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation

doi: 10.1128/JVI.01136-17

Figure Lengend Snippet: Influence of NiV-N on the STAT nuclear import system. (A) The associations between STAT1 and Impα5, Impα6, and Impα7 in the presence of N protein were evaluated by immunoprecipitation (IP). The myc-tagged importins were precipitated with an anti-myc antibody, and the coprecipitation of pSTAT1 with Impα5, Impα6, or Impα7 was evaluated in the presence or absence of N protein. A myc-tagged Impα1 construct was employed as a negative control. P protein served as a positive-control antagonist of the interaction between Impα5 and STAT1. (B) The interaction between NiV-N and HA-Impβ1 or HA-Ran was investigated with immunoprecipitation assays using anti-HA and anti-NiV-N antibodies. The myc-Impα5, myc-Impβ1, and NiV-P proteins were included as positive controls for immunoprecipitation. (C) 293T cells were transfected with pCAGGS-NiV-N (encoding a C-terminal HA tag). After 24 h, the cells were treated with 1,000 U/ml IFN-α for 30 min, and immunoprecipitation was conducted using anti-HA antibody. STAT1, STAT2, STAT3, and N protein in the lysates were detected with anti-STAT1 (E-23), -2 (C-20), and -3 (H-190) and anti-N protein antibodies, respectively. The P protein served as a positive control for an N-binding protein. For panels A to C, the experiments were independently repeated three times, and representative blots are displayed. (D) A reporter assay was conducted using 293T cells transfected with the NiV-N or N-S451A plasmid. The expression levels of each N protein and GAPDH are shown. Error bars indicate standard deviations. n.s., not significant. N-S451A-expressing Cos7 cells were treated with 2,000 U/ml of IFN-α. N-S451A and STAT1 were detected with specific antibodies and are shown as z-stack immunofluorescence images. Arrowheads and arrows indicate an N-S451A-expressing and a non-N-S451A-expressing cell, respectively. The experiment was independently conducted three times. (E) The expression plasmids for NiV-N and EGFP-Kir/Gem-W268G (referred to here as rKir/Gem) were transfected into Cos7 cells, and NiV-N was detected with an anti-NiV-N polyclonal antibody. The nuclei were stained with Hoechst dye. Images shown are z-stack data. The arrowheads and arrows point to a NiV-N-expressing and a non-NiV-N-expressing cell, respectively. The bar graph indicates the statistical evaluation of the rKir/Gem distribution. The scores were determined by counting approximately 60 cells from five randomly selected fields. n.s., not significant.

Article Snippet: The following antibodies were purchased: rabbit antibodies against STAT1 p84/p91 (E-23 [sc-346]; Santa Cruz), phospho-STAT1 (Tyr701) (9171; Cell Signaling Technology), STAT2 (C-20 [sc-476]; Santa Cruz), p-Stat2 (Tyr690) (sc-21689-R; Santa Cruz), STAT3 (H-190 [sc-7179]; Santa Cruz), histone H3 (D1H2) (4499P; Cell Signaling Technology), tubulin (H300 [sc-5546]; Santa Cruz), PKR (D-20 [sc-708]; Santa Cruz), HA tag (631207; Clontech), and c-Myc tag (C3956; Sigma-Aldrich); a rabbit IgG isotype control antibody (ab37415-5; Abcam); and mouse antibodies against STAT1α p91 (C-111 [sc-417]; Santa Cruz), STAT2 (A-7 [sc-1668] and A-9 [sc-166201]; Santa Cruz), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (MAB374; Millipore), HA tag (H9654; Sigma-Aldrich), and c-Myc (631206; Clontech).

Techniques: Immunoprecipitation, Construct, Negative Control, Positive Control, Transfection, Binding Assay, Reporter Assay, Plasmid Preparation, Expressing, Immunofluorescence, Staining

Journal: Journal of Virology

Article Title: Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation

doi: 10.1128/JVI.01136-17

Figure Lengend Snippet: Analysis of the mechanism by which NiV-N prevents STAT nuclear accumulation. (A) HeLa cells were treated with 20 nM LMB at 37°C for 1 h or 16 h, and STAT2 was detected by use of an anti-STAT2 polyclonal antibody (C-20). (B) HeLa cells were transfected with a NiV-N expression vector. At 24 h posttransfection, the cells were left untreated or treated with 20 nM LMB at 37°C for 1 h. After treatment with 1,000 U/ml IFN-α, the localization of STAT2 and N protein was investigated by use of anti-STAT2 (C-20) and anti-N protein antibodies. Arrowheads indicate cytoplasmic STAT2 in a NiV-N-expressing cell. Images shown are z-stack data. The experiment was repeated three times independently. (C) 293T cells transfected with the pGAS-Luc plasmid and the pCAGGS-NiV-N or -mP plasmid were treated with 1,000 U/ml IFN-γ, and the reporter gene activity was measured. **, P < 0.01. The expression of each protein in the cell lysates was verified by Western blot analysis. The experiment was independently repeated three times in triplicate. (D) 293T cells were transfected with pCAGGS-NiV-N (encoding a C-terminal HA tag) and -mP, and immunoprecipitation was conducted twice independently with an anti-N protein antibody. (E) The colocalization of N protein with mP (with an N-terminal HA tag) in the cytoplasm was evaluated using immunofluorescence assays. These proteins were detected by use of anti-N protein and anti-HA antibodies, respectively. Arrowheads indicate the cytoplasmic colocalization of N and mP. Z-stack images are represented. (F) NiV-N expression plasmid-transfected or untransfected 293T cells were treated with 1,000 U/ml IFN-α or IFN-γ for 20 min. The cells were fixed with 20 mM dimethyl suberimidate for 30 min and then lysed for 15 min. The cell lysates were immediately subjected to BN-PAGE. STAT1 complexes were detected by use of an anti-pSTAT1 antibody. The association between STAT1 complexes and the NiV-N protein was examined using an anti-N protein-specific antibody. (G) STAT1, pSTAT1, NiV-N, and GAPDH in the cell lysates were detected by SDS-PAGE. For panels F and G, the experiments were repeated three times, and representative data are displayed.

Article Snippet: The following antibodies were purchased: rabbit antibodies against STAT1 p84/p91 (E-23 [sc-346]; Santa Cruz), phospho-STAT1 (Tyr701) (9171; Cell Signaling Technology), STAT2 (C-20 [sc-476]; Santa Cruz), p-Stat2 (Tyr690) (sc-21689-R; Santa Cruz), STAT3 (H-190 [sc-7179]; Santa Cruz), histone H3 (D1H2) (4499P; Cell Signaling Technology), tubulin (H300 [sc-5546]; Santa Cruz), PKR (D-20 [sc-708]; Santa Cruz), HA tag (631207; Clontech), and c-Myc tag (C3956; Sigma-Aldrich); a rabbit IgG isotype control antibody (ab37415-5; Abcam); and mouse antibodies against STAT1α p91 (C-111 [sc-417]; Santa Cruz), STAT2 (A-7 [sc-1668] and A-9 [sc-166201]; Santa Cruz), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (MAB374; Millipore), HA tag (H9654; Sigma-Aldrich), and c-Myc (631206; Clontech).

Techniques: Transfection, Expressing, Plasmid Preparation, Activity Assay, Western Blot, Immunoprecipitation, Immunofluorescence, SDS Page

Journal: Journal of Virology

Article Title: Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation

doi: 10.1128/JVI.01136-17

Figure Lengend Snippet: Influence of N protein on expression of ISGs. (A) 293T cells were treated with 1,000 U/ml IFN-α in the presence or absence of NiV-N protein. The component proteins were fractionated into nuclear and cytoplasmic fractions. The whole-cell lysate and fractionated samples were subjected to SDS-PAGE to examine if the NiV-N protein decreased the nuclear pSTAT1 level. The relative nuclear STAT1 level (pSTAT1/histone H3) was measured by densitometric analysis using ImageJ software, and mean values for three independent experiments are shown. Error bars indicate standard deviations. (B) 293T cells were transfected with a NiV-N expression plasmid or empty vector, and a chromatin immunoprecipitation assay was carried out with anti-STAT1 antibody (E-23) or control IgG (ab37415-5) after treatment with 1,000 U/ml IFN-α. Enrichment of ISG promoters (IFIT2, MX2, OAS1, and PKR) was measured by quantitative PCR, and the amount of precipitated DNA relative to the amount of input DNA is shown as a percentage of the input. The experiment was repeated three times independently, and error bars show standard deviations. *, P < 0.05. (C) Expression of mRNAs of ISGs 8 h (IFIT2, OAS2, and PKR), 16 h (MX2 and OAS1), and 24 h (IFIT1 and MX1) after IFN-α treatment of 293T cells, with or without NiV-N expression, was measured by quantitative RT-PCR. GAPDH mRNA was detected as an internal control. Values shown are representative of three independent experiments. Error bars indicate standard deviations. *, P < 0.05; **, P < 0.01. (D) Expression plasmids for antagonists (pCAGGS-N and -P and pCMV-myc-V and -W) were transfected into 293T cells, and the protein expression level of PKR 16 h after IFN-α treatment was analyzed by Western blotting. PKR was detected by use of an anti-PKR antibody. Densitometric analysis of the protein bands was conducted by use of ImageJ software, and the relative PKR level (PKR/GAPDH) was calculated. Mean values for two independent experiments are shown. Error bars indicate standard deviations.

Article Snippet: The following antibodies were purchased: rabbit antibodies against STAT1 p84/p91 (E-23 [sc-346]; Santa Cruz), phospho-STAT1 (Tyr701) (9171; Cell Signaling Technology), STAT2 (C-20 [sc-476]; Santa Cruz), p-Stat2 (Tyr690) (sc-21689-R; Santa Cruz), STAT3 (H-190 [sc-7179]; Santa Cruz), histone H3 (D1H2) (4499P; Cell Signaling Technology), tubulin (H300 [sc-5546]; Santa Cruz), PKR (D-20 [sc-708]; Santa Cruz), HA tag (631207; Clontech), and c-Myc tag (C3956; Sigma-Aldrich); a rabbit IgG isotype control antibody (ab37415-5; Abcam); and mouse antibodies against STAT1α p91 (C-111 [sc-417]; Santa Cruz), STAT2 (A-7 [sc-1668] and A-9 [sc-166201]; Santa Cruz), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (MAB374; Millipore), HA tag (H9654; Sigma-Aldrich), and c-Myc (631206; Clontech).

Techniques: Expressing, SDS Page, Software, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: Kidney & blood pressure research

Article Title: Cylindromatosis (CYLD), a Deubiquitinase, Attenuates Inflammatory Signaling Pathways by Activating Toll-Like Receptor 3 in Human Mesangial Cells.

doi: 10.1159/000485084

Figure Lengend Snippet: Fig. 2. Poly IC-induced expression of RIG-I and MDA5 mRNAs was decreased but the corresponding proteins were increased by CYLD silencing.Cells were transfected with siRNA against CYLD and incubated for 48 h as in Fig. 1. Cells were then treated with 30 μg/mL poly IC. (A) After 16 h incubation, RNA was extracted from the cells and quantitative real-time RT-PCR was performed (n=3, *p<0.01). (B) After incubation for 6 h (for STAT1 and p-STAT1) or 24 h (for RIG-I and MDA5), the cells were lysed and expression of CYLD, STAT1, p-STAT1, RIG-I, MDA5, and actin was determined by western blotting. The intensity of the bands was quantified using image J software, and normalized with actin.

Article Snippet: Mouse IgG against anti-phosphorylated STAT1 (signal transducers and activator of transcription protein 1) (p-STAT1) (sc-136229) and rabbit anti-STAT1 (sc-592) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Transfection, Incubation, Quantitative RT-PCR, Western Blot, Software