Journal: Mediators of Inflammation

Article Title: Expression of the JAK/STAT Signaling Pathway in Bullous Pemphigoid and Dermatitis Herpetiformis

doi: 10.1155/2017/6716419

Figure Lengend Snippet: Immunoexpression of JAK/STAT proteins in epidermis, DH, 400x, immunohistochemistry. Immunoexpression of JAK3 in epidermis (a) perilesional skin 14.38 ± 3.61 and (e) skin lesions 18.89 ± 4.67, p > 0.05; immunoexpression of STAT2 in epidermis (b) perilesional skin 15.79 ± 2.06 and (f) skin lesions 17.15 ± 2.81. NS; immunoexpression of STAT4 in epidermis (c) perilesional skin 24.10 ± 3.40 and (g) skin lesions 29.08 ± 4.38, p < 0.05; immunoexpression of STAT6 in epidermis (d) perilesional skin 18.21 ± 3.49 and (h) skin lesions 27.85 ± 4.68, p < 0.05.

Article Snippet: Endogenous peroxidase activity was blocked by 0,3% hydrogen peroxide in distilled water, and then sections were rinsed with Tris-buffered saline (TBS, Dako, Denmark) and incubated with primary rabbit polyclonal antibody against STAT2 (Santa Cruz Biotechnology Inc.), mouse monoclonal antibody against STAT4 (Santa Cruz Biotechnology Inc.), and primary rabbit polyclonal antibody against STAT6 (Santa Cruz Biotechnology Inc.) and incubated overnight with mouse monoclonal antibody against JAK3.

Techniques: Immunohistochemistry

Journal: Mediators of Inflammation

Article Title: Expression of the JAK/STAT Signaling Pathway in Bullous Pemphigoid and Dermatitis Herpetiformis

doi: 10.1155/2017/6716419

Figure Lengend Snippet: Immunoexpression of JAK/STAT proteins in epidermis, BP, 400x, immunohistochemistry. Immunoexpression of JAK3 in epidermis (a) perilesional skin 12.54 ± 2.99 and (e) skin lesions 18.19 ± 5.58, p > 0.05; immunoexpression of STAT2 in epidermis (b) perilesional skin 14.01 ± 2.38 and (f) skin lesions 17.32 ± 2.69, p > 0.05; immunoexpression of STAT4 in epidermis (c) perilesional skin 21.05 ± 2.91 and (g) skin lesions 25.13 ± 3.56, NS; immunoexpression of STAT6 in epidermis (d) perilesional skin 17.46 ± 2.41 and (h) skin lesions 26.09 ± 4.45, NS.

Article Snippet: Endogenous peroxidase activity was blocked by 0,3% hydrogen peroxide in distilled water, and then sections were rinsed with Tris-buffered saline (TBS, Dako, Denmark) and incubated with primary rabbit polyclonal antibody against STAT2 (Santa Cruz Biotechnology Inc.), mouse monoclonal antibody against STAT4 (Santa Cruz Biotechnology Inc.), and primary rabbit polyclonal antibody against STAT6 (Santa Cruz Biotechnology Inc.) and incubated overnight with mouse monoclonal antibody against JAK3.

Techniques: Immunohistochemistry

Journal: Mediators of Inflammation

Article Title: Expression of the JAK/STAT Signaling Pathway in Bullous Pemphigoid and Dermatitis Herpetiformis

doi: 10.1155/2017/6716419

Figure Lengend Snippet: Immunoexpression of JAK/STAT proteins in epidermis, normal skin, 400x, immunohistochemistry. (a) Immunoexpression of JAK3 in epidermis, normal skin, 10.73 ± 3.36. (b) Immunoexpression of STAT2 in epidermis, normal skin, 11.06 ± 5.34. (c) Immunoexpression of STAT4 in epidermis, normal skin, 18.59 ± 3.01. (d) Immunoexpression of STAT6 in epidermis, normal skin, 11.56 ± 2.84.

Article Snippet: Endogenous peroxidase activity was blocked by 0,3% hydrogen peroxide in distilled water, and then sections were rinsed with Tris-buffered saline (TBS, Dako, Denmark) and incubated with primary rabbit polyclonal antibody against STAT2 (Santa Cruz Biotechnology Inc.), mouse monoclonal antibody against STAT4 (Santa Cruz Biotechnology Inc.), and primary rabbit polyclonal antibody against STAT6 (Santa Cruz Biotechnology Inc.) and incubated overnight with mouse monoclonal antibody against JAK3.

Techniques: Immunohistochemistry

Journal: Mediators of Inflammation

Article Title: Expression of the JAK/STAT Signaling Pathway in Bullous Pemphigoid and Dermatitis Herpetiformis

doi: 10.1155/2017/6716419

Figure Lengend Snippet: Morphometric analysis of JAK3 (a), STAT2 (b), STAT4 (c), and STAT6 (d) immunoexpression in keratinocytes, evaluated with immunohistochemistry. The results of semiquantitative analysis are expressed as the mean ± SD. Control—normal skin. DH—dermatitis herpetiformis, skin lesions. BP—bullous pemphigoid, skin lesions. The level of significance is defined where p < 0.05. JAK3: C versus DH ( p < 0.05), C versus BP ( p < 0.05), and DH versus BP (NS). STAT2: C versus DH ( p < 0.05), C versus BP ( p < 0.05), and BP versus DH (NS). STAT4: C versus DH ( p < 0.05), C versus BP ( p < 0.05), and DH versus BP ( p < 0.05). STAT6: C versus DH ( p < 0.05), C versus BP ( p < 0.05), and DH versus BP (NS).

Article Snippet: Endogenous peroxidase activity was blocked by 0,3% hydrogen peroxide in distilled water, and then sections were rinsed with Tris-buffered saline (TBS, Dako, Denmark) and incubated with primary rabbit polyclonal antibody against STAT2 (Santa Cruz Biotechnology Inc.), mouse monoclonal antibody against STAT4 (Santa Cruz Biotechnology Inc.), and primary rabbit polyclonal antibody against STAT6 (Santa Cruz Biotechnology Inc.) and incubated overnight with mouse monoclonal antibody against JAK3.

Techniques: Immunohistochemistry, Control

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-23 Mediates Osteoclastogenesis in Collagen-Induced Arthritis by Modulating MicroRNA-223

doi: 10.3390/ijms23179718

Figure Lengend Snippet: Endogenous expression of IL-23p19 and clinical assessments in the LVshIL-23p19-treated mice with collagen-induced arthritis (CIA). ( A ) Reverse transcription polymerase chain reaction (RT-PCR) analysis of IL-23p19 expression in the synovial tissue of mice with CIA (Day 10, 21, 42). Each lane represents pooled samples from 3 animals. Results are representative of two independent experiments. ( B ) Amelioration in mice with CIA by evaluating with arthritis score after the LVshIL-23p19 treatment. Mice that had been immunized with collagen on days 0 and 21 were injected intraperitoneally with LVshIL-23p19, LVshLuc and medium on day 21, respectively. The arrow indicates the time at which the lentiviral vectors were injected. Each value shown represents the mean ± SEM ( n = 6). ( C ) Representative images of the ankle sections by hematoxylin and eosin staining on day 45. ( D ) Representative images and quantification of the levels of phosphorylated signal transducers and activators of transcription 4 (p-STAT4) by immunohistochemical staining on day 45. Each value shown represents the mean ± SEM ( n = 3). ( E ) Representative images of the levels of miR-223 by in situ hybridization on day 45. Scale bars represent 500, 200 and 50 μm in ×40, ×100 and ×400 magnifications, respectively. Each value shown represents the mean ± SEM ( n = 3). Black boxed areas were shown at higher magnification in the panels (×400) beneath them. Results are representative of two independent experiments.

Article Snippet: Briefly, IL-23-treated RAW264.7 cells were formaldehyde crossed-linked, and cell lysates were sonicated to shear the DNA to an average length of 500 bp followed by immunoprecipitation with rabbit anti-phosphorylated STAT4 (ChIP grade, Cell Signaling Technology, Danvers, MA, USA) antibody and normal rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) in combination with protein G agarose beads.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, Staining, Immunohistochemical staining, In Situ Hybridization

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-23 Mediates Osteoclastogenesis in Collagen-Induced Arthritis by Modulating MicroRNA-223

doi: 10.3390/ijms23179718

Figure Lengend Snippet: Association of IL-23 signaling with miR-223 in osteoclastogenesis. ( A ) Tartrate-resistant acid phosphatase (TRAP) staining and quantification in IL-23 (5 and 20 ng/mL) treated bone marrow-derived macrophages (BMMs). Each value shown represents the mean ± SEM ( n = 4). ( B ) TRAP staining and quantification in LVmiR-223T and LVshLuc-infected BMMs (MOI = 0.05 and 0.5) in response to IL-23 stimulation (20 ng/mL). Each value shown represents the mean ± SEM ( n = 3). ( C ) TRAP staining and quantification in LVshSTAT4#1 (TRCN0000081638), LVshSTAT4#2 (TRCN0000081639) and LVshLuc-infected BMMs (MOI = 1 and 5) in response to IL-23 stimulation (20 ng/mL). Scale bars represent 200 μm in ×100 magnifications. Each value shown represents the mean ± SEM ( n = 3). ( D ) Binding of phosphorylated STAT4 to the miR-223 promoter in IL-23-treated RAW264.7 cells by determining with quantitative chromatin immunoprecipitation (qChIP) assay. Each value shown represents the mean ± SEM ( n = 3). ( E ) Expression of miR-223 in BMMs treated with various concentrations of IL-23 for 12 h, as determined by quantitative RT-PCR (qRT-PCR). Each value shown represents the mean ± SEM ( n = 3). ( F ) Expression of miR-223 in LVshLuc, LVshSTAT4#1 (TRCN0000081638) and LVshSTAT4#2 (TRCN0000081639)-transduced BMMs upon stimulating with IL-23 (20 ng/mL) for 12 h, as determined by qRT-PCR. Each value shown represents the mean ± SEM ( n = 3). Red boxed areas are shown at higher magnification in the panels beneath them. Results are representative of two independent experiments.

Article Snippet: Briefly, IL-23-treated RAW264.7 cells were formaldehyde crossed-linked, and cell lysates were sonicated to shear the DNA to an average length of 500 bp followed by immunoprecipitation with rabbit anti-phosphorylated STAT4 (ChIP grade, Cell Signaling Technology, Danvers, MA, USA) antibody and normal rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) in combination with protein G agarose beads.

Techniques: Staining, Derivative Assay, Infection, Binding Assay, Chromatin Immunoprecipitation, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-23 Mediates Osteoclastogenesis in Collagen-Induced Arthritis by Modulating MicroRNA-223

doi: 10.3390/ijms23179718

Figure Lengend Snippet: Lisofylline (LSF) treatment in BMMs and mice with CIA. ( A ) TRAP staining and quantification in LSF (50 and 100 μM)-treated BMMs upon stimulating with IL-23 (20 ng/mL). Each value shown represents the mean ± SEM ( n = 3). Red boxed areas are shown at higher magnification in the panels next to them. Scale bars represent 200 μm in ×100 magnifications. ( B ) Expression of miR-223 in BMMs treated with various concentrations of LSF in response to IL-23 (20 ng/mL) stimulation for 12 h, as determined by qRT-PCR. Each value shown represents the mean ± SEM ( n = 3). ( C ) Arthritis score and incidence in mice with CIA through intraperitoneal injections of LSF (50 mg/kg) and PBS daily from day 22 to 36. Arrows indicate the time at which LSF was injected. Each value shown represents the mean ± SEM (PBS: n = 6, LSF: n = 6). ( D ) Representative images of the ankle sections by hematoxylin and eosin staining on day 40. Scale bars represent 200 and 50 μm in ×100 and ×400 magnifications, respectively. Each value shown represents the mean ± SEM ( n = 6). Black boxed areas were shown at higher magnification in the panels (×400) beneath them. Expression of ( E ) miR-223, ( F ) phospho-STAT4 (p-STAT4) and ( G ) the number of TRAP-positive cells in joint extracts of LSF and PBS-injected mice with CIA, as determined by qRT-PCR. Each value shown represents the mean ± SEM ( n = 6). Results are representative of two independent experiments.

Article Snippet: Briefly, IL-23-treated RAW264.7 cells were formaldehyde crossed-linked, and cell lysates were sonicated to shear the DNA to an average length of 500 bp followed by immunoprecipitation with rabbit anti-phosphorylated STAT4 (ChIP grade, Cell Signaling Technology, Danvers, MA, USA) antibody and normal rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) in combination with protein G agarose beads.

Techniques: Staining, Expressing, Quantitative RT-PCR, Injection

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-23 Mediates Osteoclastogenesis in Collagen-Induced Arthritis by Modulating MicroRNA-223

doi: 10.3390/ijms23179718

Figure Lengend Snippet: A schematic overview of silencing IL-23-STAT4-miR-223 signaling axis in preventing osteoclastogenesis. IL-23p19 can bind to the IL-23 receptor (IL-23R), which causes phosphorylation and translocation of STAT4 into the nucleus. P-STAT4 further conjugates with the promoter region of primary miR-223 (pri-miR-223) and the transactivated pri-miR-223 translocates into the cytosol to become mature miR-223 (miR-223) and mediates osteoclastogenesis. The signaling axis, IL-23-STAT4-miR-223 is proved to mediate osteoclastogenesis by introducing IL-23-stimulated BMMs and mouse CIA model with LVshIL-23p19, LVshSTAT4, LSF, and LVmiR-223T that target IL-23p19, STAT4, and miR-223.

Article Snippet: Briefly, IL-23-treated RAW264.7 cells were formaldehyde crossed-linked, and cell lysates were sonicated to shear the DNA to an average length of 500 bp followed by immunoprecipitation with rabbit anti-phosphorylated STAT4 (ChIP grade, Cell Signaling Technology, Danvers, MA, USA) antibody and normal rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) in combination with protein G agarose beads.

Techniques: Translocation Assay

Journal: Cell reports

Article Title: Mass Cytometry Reveals Global Immune Remodeling with Multi-lineage Hypersensitivity to Type I Interferon in Down Syndrome

doi: 10.1016/j.celrep.2019.10.038

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-human phospho-STAT4 (Tyr693) (clone 38/p-Stat4) , Fluidigm , Cat#3148006A; RRID:AB_2811098.

Techniques: Recombinant, Mass Cytometry, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: Cynaropicrin Shows Antitumor Progression Potential in Colorectal Cancer Through Mediation of the LIFR/STATs Axis

doi: 10.3389/fcell.2020.605184

Figure Lengend Snippet: Cynaropicrin stimulation inhibits the formation of STAT3/STAT4 heterodimers activated by LIFR. (A,B) The cynaropicrin molecular docking at the contact interface between LIFR and LIF resulted in 20 conformations. Energy decomposition analysis based on residues suggests that GLN209 may be the key residue to maintain binding. (C) Confirmation of LIFR- Cynaropicrin interaction. LIFR Protein captured on NTA chip can bind Cynaropicrin, with an affinity constant of 134 μM as determined in a SPR assay. (D,E) RKO cells were treated with (+) or without (-)cynaropicrin for 24 h, and then total cell protein was extracted to perform a co-immunoprecipitation assay using STAT3 antibody or STAT4 antibody. The immunoprecipitation complexes were further separated by Western blotting. (F) RKO cells were pretreated for 24 h and then stimulated with IL-6 (25 ng/mL) for 30 min. Subcellular localization of STAT4 was detected by immunofluorescence staining and laser confocal microscopy.

Article Snippet: The following primary antibodies were commercially obtained: anti-GAPDH (AB-P-R 001, GoodHere Technology), anti-STAT3 (phospho Y705) (ab76315, Abcam), STAT3 mAb (#12640, Cell Signaling Technology), Phospho-STAT4 (Tyr693) mAb (#4134, Cell Signaling Technology), STAT4 mAb (#2653, Cell Signaling Technology), anti-LIFR (sc-515337, Santa Cruz, CA, United States), anti-LIF (sc-515931, Santa Cruz, CA, United States), anti-Bax (ab32503, Abcam), anti-Bcl-2 (sc-56015, Santa Cruz, CA, United States), and Anti-Cleaved PARP1 (ab32064, Abcam).

Techniques: Residue, Binding Assay, SPR Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Confocal Microscopy

Journal: Infection and Immunity

Article Title: TRP75-mediated STAT3 activation promotes anti-apoptotic signaling and Ehrlichia chaffeensis infection

doi: 10.1128/iai.00459-25

Figure Lengend Snippet: Nuclear translocation of STAT family members during E. chaffeensis infection. ( A ) Uninfected or E. chaffeensis -infected THP-1 cells at 72 hpi (MOI 10) were probed for STAT1–6 (green) and E. chaffeensis marker Dsb (red) by immunofluorescent microscopy. 4´,6-Diamidino-2-phenylindole (DAPI) (blue) was used to stain nuclei. Scale bar = 20 µm. ( B ) Nuclear regions of interest (ROIs) were defined by DAPI signal, and mean nuclear STAT3 intensity per cell was quantified using ImageJ. Statistical significance was determined using two-tailed Student’s t -test; * P < 0.05, ** P < 0.01. Data represent the mean ± SD of three independent biological replicates ( n = 3), and representative images are shown.

Article Snippet: The primary antibodies utilized in this research include monoclonal rabbit α-STAT1 (14994S; Cell Signaling Technology), monoclonal rabbit α-STAT2 (72604S; Cell Signaling Technology), rabbit α-STAT3 (12640S; Cell Signaling Technology), monoclonal rabbit α-STAT4 (2653S; Cell Signaling Technology), monoclonal rabbit α-STAT5 (94205S; Cell Signaling Technology), monoclonal rabbit α-STAT6 (5397S; Cell Signaling Technology), monoclonal rabbit α-phospho-STAT3 Y705 (9145S; Cell Signaling Technology), monoclonal rabbit α-MCL-1 (94296S; Cell Signaling Technology), monoclonal rabbit α-caspase 3 (9662S; Cell Signaling Technology), monoclonal mouse α-caspase 7 (9494S; Cell Signaling Technology), monoclonal mouse α-GAPDH (sc-47724; Santa Cruz Biotechnology), monoclonal mouse α-Tubulin (sc-5286; Santa Cruz Biotechnology), monoclonal mouse α-Vinculin (sc-73614; Santa Cruz Biotechnology), monoclonal mouse α-GFP (sc-9996; Santa Cruz Biotechnology), rabbit α-disulfide bond formation protein (Dsb) , and rabbit α-TRP75 ( ).

Techniques: Translocation Assay, Infection, Marker, Microscopy, Staining, Two Tailed Test