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:

Article Title: Association of Mumps Virus V Protein with RACK1 Results in Dissociation of STAT-1 from the Alpha Interferon Receptor Complex

doi: 10.1128/JVI.76.24.12676-12682.2002

Figure Lengend Snippet: Association between RACK1 and MuV-V in MuV-infected cells. (A) Whole-cell lysates of FL cells (lane 1) and FLMT cells (lanes 2 and 3) were immunoprecipitated (IP) with anti-V protein antibody (lanes 1 and 3) or preimmune serum (Pre) (lane 2) and detected by Western blot (WB) analysis with anti-RACK1 antibody (upper panel) or anti-V protein antibody (lower panel). (B) FLMT cells were treated (+) or not treated (−) with IFN for 15 min, and then cell lysates were immunoprecipitated with anti-V protein antibody (V) (lanes 1 and 2) or anti-RACK1 antibody (R) (lanes 3 and 4) and subjected to immunoblotting with anti-RACK1 antibody (upper panel) or anti-V protein antibody (lower panel). (C) FL cells (lanes 1 and 2) and FLMT cells (lanes 3 and 4) were treated (+) or not treated (−) with IFN for 15 min, and then cell lysates were immunoprecipitated with anti-RACK1 antibody and analyzed by Western blotting with anti-STAT-1 antibody (upper panel) or anti-V protein antibody (lower panel). (D) FL cells (lanes 1 and 2) and FLMT cells (lanes 3 and 4) were treated (+) or not treated (−) with IFN for 24 h, and then the levels of production of RACK1 (upper panel) and MuV-V (lower panel) were investigated by immunoblotting.

Article Snippet: Rabbit polyclonal antibodies against STAT-1 p84/p91 (E-23) and STAT-2 p133 (C-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Infection, Immunoprecipitation, Western Blot

Journal:

Article Title: Association of Mumps Virus V Protein with RACK1 Results in Dissociation of STAT-1 from the Alpha Interferon Receptor Complex

doi: 10.1128/JVI.76.24.12676-12682.2002

Figure Lengend Snippet: Effect of MuV infection on the association between IFN-αRβL and RACK1. A GST fusion protein containing the entire cytoplasmic domain of IFN-αRβLc (βL) was used to pull down RACK1 (lanes 2 and 3), STAT-1 (lanes 5 and 6), STAT-2 (lanes 8 and 9), and MuV-V (lane 11) from lysates obtained from Akata cells (A) (lanes 1, 2, 4, 5, 7, and 8) or Akata-MP cells (A-MP) (lanes 3, 6, 9, 10, and 11). GST alone was used as a negative control. WB, Western blotting. Sup(1/50), 1/50 volume of supernatant after pull-down experiment.

Article Snippet: Rabbit polyclonal antibodies against STAT-1 p84/p91 (E-23) and STAT-2 p133 (C-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Infection, Negative Control, Western Blot

Journal:

Article Title: Association of Mumps Virus V Protein with RACK1 Results in Dissociation of STAT-1 from the Alpha Interferon Receptor Complex

doi: 10.1128/JVI.76.24.12676-12682.2002

Figure Lengend Snippet: Influence of MuV-V on the association between RACK1 and STAT-1 in vitro. (A) His6-tagged STAT-1 (lanes 1, 2, and 4), GST-fused MuV-V (lanes 2 and 3), GST-fused MuV-Vsp (lanes 4 and 5), and GST-fused RACK1 (lanes 1 to 5) were mixed as shown and coimmunoprecipitated (IP) with anti-RACK1 antibody. Precipitates were subjected to immunoblotting (Western blotting [WB]) with anti-STAT-1, anti-MuV-V, and anti-RACK1 antibodies. (B and C) His6-tagged STAT-1, GST-fused RACK1, GST-fused MuV-V, and GST-fused Vc189a were mixed as shown and coimmunoprecipitated with anti-STAT-1 antibody (B) or anti-MuV-V antibody (C). RACK1 and STAT-1 in the precipitates were investigated by Western blotting analysis with anti-RACK1 antibody or anti-STAT-1 antibody.

Article Snippet: Rabbit polyclonal antibodies against STAT-1 p84/p91 (E-23) and STAT-2 p133 (C-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: In Vitro, Western Blot

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: Acta Biochimica et Biophysica Sinica

Article Title: SPATS2L is a positive feedback regulator of the type I interferon signaling pathway and plays a vital role in lupus

doi: 10.3724/abbs.2024132

Figure Lengend Snippet: SPATS2L influences the expression level and phosphorylation of STAT1 within the type I IFN signaling pathway (A) The dual-luciferase reporter assay suggested that SPATS2L may regulate the IFN signaling pathway by enhancing the activity of ISRE. Attenuation of SPATS2L expression results in a decrease in ISRE activity. Notably, this reduction was particularly conspicuous after the induction of the IFN signaling pathway (IFN-α, 1000 U/mL, 6 h). (B) SPATS2L affects the expression level of the key molecule STAT1 in the IFN signaling pathway. RT-qPCR revealed that the mRNA levels of STAT1 and JAK1 were significantly attenuated after SPATS2L knockdown, regardless of whether the IFN signaling pathway was activated. (C) The RT-qPCR results also indicated that overexpression of SPATS2L led to a significant increase in the expression of both STAT1 and JAK1. Data are presented as the mean±SEM of three independent experiments, and the P values were analyzed using a two-tailed unpaired t-test. (D) SPATS2L knockdown significantly reduced the phosphorylation of STAT1. The Jurkat cells in the KD and NC groups were stimulated with IFN-α (1000 U/mL) for 15 and 60 min, respectively. Subsequently, we extracted proteins for western blot analysis. (E,F) Imaging flow cytometry revealed that the downregulation of SPATS2L expression had an impact on the nuclear translocation efficiency of STAT1. After SPATS2L was knocked down in Jurkat cells and stimulated with IFN-α (1000 U/mL) for 60 min, the nuclear translocation efficiency was significantly reduced compared to that in the NC group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: The antibody ( Supplementary Table S3 ) used in the imaging flow cytometry experiment was a rabbit monoclonal antibody against STAT1 (PE Conjugate; Cell Signaling Technology).

Techniques: Expressing, Phospho-proteomics, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, Knockdown, Over Expression, Two Tailed Test, Western Blot, Imaging, Flow Cytometry, Translocation Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: SPATS2L is a positive feedback regulator of the type I interferon signaling pathway and plays a vital role in lupus

doi: 10.3724/abbs.2024132

Figure Lengend Snippet: Type I IFNs regulate the expression of SPATS2L (A) ChIP-seq data from the K562 cell line obtained from the ENCODE database indicated the presence of binding peaks for STAT1, STAT2, and IRF9 in the promoter region of SPATS2L. Additionally, an ISRE motif sequence was found within the promoter region of SPATS2L. (B,C) PBMCs from four healthy individuals were treated with different concentrations (0, 50, 200, or 1000 ng) of IFN-α and divided into various time groups (0, 3, 6, or 12 h) of stimulation. SPATS2L was induced by IFN-α in a concentration-dependent manner. (D) IFN-α induces the expression of SAST2L in Jurkat cells in a concentration-dependent manner. Data are presented as the mean±SEM of four independent experiments, and the P values were analyzed using a two-tailed unpaired t test. (E,F) In the PBMCs of healthy volunteers, after 4 h of stimulation with 100 ng/mL IFN-α, the expression level of SPATS2L was significantly increased in various cell subpopulations, including DCs, CD8+ T cells, CD4+ T cells, monocytes, and NK cells. (G) Compared with that in patients receiving placebo treatment, the expression of SPATS2L in patients with SLE was significantly lower after 36 weeks of IFN-K treatment. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: The antibody ( Supplementary Table S3 ) used in the imaging flow cytometry experiment was a rabbit monoclonal antibody against STAT1 (PE Conjugate; Cell Signaling Technology).

Techniques: Expressing, ChIP-sequencing, Binding Assay, Sequencing, Concentration Assay, Two Tailed Test

Journal: Acta Biochimica et Biophysica Sinica

Article Title: SPATS2L is a positive feedback regulator of the type I interferon signaling pathway and plays a vital role in lupus

doi: 10.3724/abbs.2024132

Figure Lengend Snippet: Targeted intervention targeting the positive regulator SPATS2L within the IFN signaling pathway is beneficial for restoring the abnormally activated IFN signaling pathway in SLE patients (A) The scRNA-seq indicated that SPATS2L is highly expressed in cDCs and monocytes. Additionally, compared to that in healthy individuals, SPATS2L expression is significantly upregulated in various cell subsets of SLE patients, including CD14+ and CD16+ monocytes, CD4+ and CD8+ T cells, and B cells. (B) SPATS2L expression in single cells. (C,D) siRNA-mediated inhibition of SPATS2L gene expression in SLE patients using siRNA. After SPATS2L was knocked down, the IFN signaling pathway in patients with SLE was suppressed, leading to the significant downregulation of ISGs such as STAT1, OAS1, and IFIT2. Data are presented as the mean±SEM of four independent experiments, and the P values were analyzed using a two-tailed paired t-test. *P<0.05, **P<0.01, ***P<0.001. (E) The working model of SPATS2L-mediated positive regulation of the type I IFN signaling pathway triggering SLE flare-up. SPATS2L is an IFN-inducible gene that is highly expressed in patients with SLE, and even more so in patients with SLE. SPATS2L exerts its regulatory effects on ISGs mainly by positively modulating type I IFN signaling, thereby further promoting the progression of SLE.

Article Snippet: The antibody ( Supplementary Table S3 ) used in the imaging flow cytometry experiment was a rabbit monoclonal antibody against STAT1 (PE Conjugate; Cell Signaling Technology).

Techniques: Expressing, Inhibition, Gene Expression, Two Tailed Test