Journal: Cancer medicine

Article Title: Direct effect of dasatinib on signal transduction pathways associated with a rapid mobilization of cytotoxic lymphocytes.

doi: 10.1002/cam4.925

Figure Lengend Snippet: Figure 1. Flow cytometric analysis of each lymphocyte subset and expression of phosphorylated proteins. Representative data are shown. Lymphocyte fractions are classified according to surface antibodies including CD3, CD8, and CD56. Natural killer (NK) cells were defined as the CD3-CD56+ immunophenotype and cytotoxic T lymphocytes (CTLs) were CD3+ CD8+ (A). Cells were costained with phospho-specific antibodies, including antibodies targeting pJAK1, pJAK2, pSTAT1, pSTAT3, pERK, pJNK, pp38, and pAKT, and expression levels of the isotype control and phosphorylated proteins in NK cells are presented (B). The values for the isotype control and each phosphorylated protein are shown as the median fluorescence intensity (MFI).

Article Snippet: Fluorescein isothiocyanate (FITC)- labeled polyclonal rabbit IgG antibodies for phosphorylated proteins, including pJAK1 (Y1034, #3238R), pJAK2 (Y1007, #2485R), pSTAT1 (Y701, #1657R), pSTAT3 (Y705, #1658R), pERK (T202/Y204, #1646R), pJNK (T183/Y185, #1640R), pp38 (T180/Y182, #2210R), pAKT (Y315, #5193R), and isotype control (#0295P) were purchased from Bioss (Wobun, MA, USA).

Techniques: Expressing, Control, Fluorescence

Journal: Cancer medicine

Article Title: Direct effect of dasatinib on signal transduction pathways associated with a rapid mobilization of cytotoxic lymphocytes.

doi: 10.1002/cam4.925

Figure Lengend Snippet: Figure 3. Constitutive levels of phosphorylated proteins including pJAK1, pJAK2, pSTAT1, pSTAT3, pERK, pJNK, pp38, and pAKT in natural killer (NK) cells (A) and cytotoxic T lymphocytes (CTLs) (B) grouped according to treatment (dasatinib [n = 18] or other TKI [n = 12]) are shown. The values for phosphorylated proteins in each fraction are shown as the median fluorescence intensity (MFI).

Article Snippet: Fluorescein isothiocyanate (FITC)- labeled polyclonal rabbit IgG antibodies for phosphorylated proteins, including pJAK1 (Y1034, #3238R), pJAK2 (Y1007, #2485R), pSTAT1 (Y701, #1657R), pSTAT3 (Y705, #1658R), pERK (T202/Y204, #1646R), pJNK (T183/Y185, #1640R), pp38 (T180/Y182, #2210R), pAKT (Y315, #5193R), and isotype control (#0295P) were purchased from Bioss (Wobun, MA, USA).

Techniques: Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: P orphyromonas gingivalis promotes the progression of oral squamous cell carcinoma by activating the neutrophil chemotaxis in the tumour microenvironment

doi: 10.1007/s00262-022-03348-5

Figure Lengend Snippet: Acquisition of the EMT phenotype was verified by activation of the JAK1/STAT3 signalling pathway through the CXCL2/CXCR2 signalling axis. a Western blot analysis showed that EMT occurred and the JAK1/STAT3 signalling pathway was activated by CXCL2/CXCR2 when P. gingivalis infected the TME of OSCC. b After coculture for 24 h, and the cells were transinfected with sh-CXCL2 and sh-CXCR2 lentiviruses for 72 h, the cells were observed under a fluorescence microscope. c, d Knockdown of CXCL2 and CXCR2 was verified at the protein and mRNA levels by Western blotting and qRT-PCR

Article Snippet: Primary antibodies against P. gingivalis (Dia-An, lnc, it was freshly prepared, 1:1000), CXCL2 (bs-1162R, BIOSS, Biotech, Beijing, China, 1:2000), CXCR2 (bs-1629R, BIOSS, Biotech, Beijing, China, 1:2000), JAK1 (bs-1439R, BIOSS, Biotech, Beijing, China, 1:2000), STAT3 (bs-55208R, BIOSS, Biotech, Beijing, China, 1:2000), p-JAK1 (bs-3238R, BIOSS, Biotech, Beijing, China, 1:2000), p-STAT3 (bs-1658R, BIOSS, Biotech, Beijing, China, 1:2000), E-cadherin (bs-10009R, BIOSS, Biotech, Beijing, China, 1:2000), N-cadherin (bs-1172R, BIOSS, Biotech, Beijing, China, 1:2000), and GAPDH (bs-13282R, BIOSS, Biotech, Beijing, China, 1:2000) and secondary antibodies (anti‐rabbit‐HRP, Cell Signalling Technology, Inc, Boston, USA) were used in this study.

Techniques: Activation Assay, Western Blot, Infection, Fluorescence, Microscopy, Knockdown, Quantitative RT-PCR

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: P orphyromonas gingivalis promotes the progression of oral squamous cell carcinoma by activating the neutrophil chemotaxis in the tumour microenvironment

doi: 10.1007/s00262-022-03348-5

Figure Lengend Snippet: Screening and culture of positive clone cells expressing both the lentiviruses of sh-CXCL2 and sh-CXCR2 which can reverse the phenotype of EMT and rescue the signalling pathway of JAK1/STAT3. a The lentiviruses of sh-CXCL2 and sh-CXCR2 were co-transfected into TSCCa cells. b Flow cytometry was used to screen out the positive cells that expression both lentiviruses of sh-CXCL2 and sh-CXCR2. c The clone of positive cells were screened out and cultured by limited dilution method. d Western blot analysis after inactivation of the CXCL2/CXCR2 signalling axis in the P. gingivalis-infected TME of OSCC, the activity of the JAK1/STAT3 signalling pathway was decreased, and the EMT process was reversed. (*p < 0.05, **p < 0.01, and ***p < 0.001; one‐way analysis of variance)

Article Snippet: Primary antibodies against P. gingivalis (Dia-An, lnc, it was freshly prepared, 1:1000), CXCL2 (bs-1162R, BIOSS, Biotech, Beijing, China, 1:2000), CXCR2 (bs-1629R, BIOSS, Biotech, Beijing, China, 1:2000), JAK1 (bs-1439R, BIOSS, Biotech, Beijing, China, 1:2000), STAT3 (bs-55208R, BIOSS, Biotech, Beijing, China, 1:2000), p-JAK1 (bs-3238R, BIOSS, Biotech, Beijing, China, 1:2000), p-STAT3 (bs-1658R, BIOSS, Biotech, Beijing, China, 1:2000), E-cadherin (bs-10009R, BIOSS, Biotech, Beijing, China, 1:2000), N-cadherin (bs-1172R, BIOSS, Biotech, Beijing, China, 1:2000), and GAPDH (bs-13282R, BIOSS, Biotech, Beijing, China, 1:2000) and secondary antibodies (anti‐rabbit‐HRP, Cell Signalling Technology, Inc, Boston, USA) were used in this study.

Techniques: Expressing, Transfection, Flow Cytometry, Cell Culture, Western Blot, Infection, Activity Assay

Journal: Journal of Neuroinflammation

Article Title: IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-021-02156-5

Figure Lengend Snippet: Upregulation of IL-9 and inflammatory cytokines as well as the activation of Notch1 signaling during EAE process. a The level of IL-9 in spinal cords of EAE mice with different clinical scores was measured by real-time PCR and Western blot assay, respectively. b The expressions of GFAP, NICD and p-STAT3 in spinal cords along with EAE process were detected by Western blot assay. c The mRNA levels of IL-6, TNF-α, IP-10, and MCP-1 in spinal cords were determined using real-time PCR. d The secretion levels of IL-6, TNF-α, IP-10 and MCP-1 in the sera were detected by Cytometric Bead Array (CBA) during the EAE process. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus PBS group ( n = 6/group, one-way ANOVA). These results were repeated four times. Data are represented as the mean ± SEM

Article Snippet: Antibodies used in this study were as follows: anti-IL-9 antibody (Rabbit, ab203386, Abcam), anti-GFAP antibody (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-cleaved Notch1 antibody (NICD, Rabbit, 4147, Cell Signaling Technology), anti-Notch 1 antibody (Goat, sc-6014, SANTA CRUZ), anti-p-STAT3 (Rabbit, Tyr705, BS4181, Bioworld Technology), anti-STAT3 (Rabbit, AP0365, Bioworld Technology), NF-κB p65 (Rabbit, 8242, Cell Signaling Technology), anti- CBP/P300 antibody (Rabbit, 4771s, Cell Signaling Technology), anti-RNA Pol II antibody (Mouse, 17-620, Sigma Aldrich), and anti-H3K27ac antibody (Rabbit, ab177178, Abcam).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Neuroinflammation

Article Title: IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-021-02156-5

Figure Lengend Snippet: IL-9 activates the Notch1 pathway and promotes inflammatory cytokines production in astrocytes. Primary mouse astrocytes were incubated in a serum-free medium overnight followed by treating with IL-9 at the indicated time point. a The expression changes of GFAP, NICD, and p-STAT3 were analyzed by Western blot assay. b Immunofluorescent staining for GFAP (green), Notch1 (red), and nuclear staining of DAPI (blue) in cultured astrocytes with IL-9 treatment for 6 h. Scale bars, 50 μm. c The mRNA levels of IL-6, TNF-α, IP-10, and MCP-1 in astrocytes were detected using real-time PCR assay. d The secretion levels of IL-6, TNF-α, IP-10, and MCP-1 in the supernatant of astrocytes were measured by CBA assay. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus DMEM group (one-way ANOVA). The data are from three independent experiments and represented as the mean ± SEM

Article Snippet: Antibodies used in this study were as follows: anti-IL-9 antibody (Rabbit, ab203386, Abcam), anti-GFAP antibody (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-cleaved Notch1 antibody (NICD, Rabbit, 4147, Cell Signaling Technology), anti-Notch 1 antibody (Goat, sc-6014, SANTA CRUZ), anti-p-STAT3 (Rabbit, Tyr705, BS4181, Bioworld Technology), anti-STAT3 (Rabbit, AP0365, Bioworld Technology), NF-κB p65 (Rabbit, 8242, Cell Signaling Technology), anti- CBP/P300 antibody (Rabbit, 4771s, Cell Signaling Technology), anti-RNA Pol II antibody (Mouse, 17-620, Sigma Aldrich), and anti-H3K27ac antibody (Rabbit, ab177178, Abcam).

Techniques: Incubation, Expressing, Western Blot, Staining, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Journal of Neuroinflammation

Article Title: IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-021-02156-5

Figure Lengend Snippet: Inhibition of Gm13568 downregulates Notch1 signaling activation as well as inflammatory cytokine production in astrocytes by IL-9. a Primary mouse astrocytes were infected with recombinant lentivirus, LV-Inhibit-Gm13568 and LV-ctrl, for 72 h, respectively. Then, the astrocytes were incubated in a serum-free medium overnight followed by IL-9 stimulation for 6 h. Western blot assay was used for measuring the protein expressions of GFAP, Notch1/NICD, and p-STAT3. b The mRNA levels of IL-6, TNF-α and IP-10 in astrocytes were measured by real-time PCR assay. c The secretion of IL-6, TNF-α, and IP-10 in the supernatant of astrocytes were analyzed by CBA assay. The data are represented as the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus DMEM group; ## p < 0.01 and ### p < 0.001 versus LV-ctrl+IL-9 group (one-way ANOVA, the two-tailed Student’s t test)

Article Snippet: Antibodies used in this study were as follows: anti-IL-9 antibody (Rabbit, ab203386, Abcam), anti-GFAP antibody (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-cleaved Notch1 antibody (NICD, Rabbit, 4147, Cell Signaling Technology), anti-Notch 1 antibody (Goat, sc-6014, SANTA CRUZ), anti-p-STAT3 (Rabbit, Tyr705, BS4181, Bioworld Technology), anti-STAT3 (Rabbit, AP0365, Bioworld Technology), NF-κB p65 (Rabbit, 8242, Cell Signaling Technology), anti- CBP/P300 antibody (Rabbit, 4771s, Cell Signaling Technology), anti-RNA Pol II antibody (Mouse, 17-620, Sigma Aldrich), and anti-H3K27ac antibody (Rabbit, ab177178, Abcam).

Techniques: Inhibition, Activation Assay, Infection, Recombinant, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis

doi: 10.1186/s12974-021-02156-5

Figure Lengend Snippet: Knockdown of Notch1 in astrocytes suppresses inflammation and alleviates EAE in mice. Mice were subjected to recombinant lentiviruses, LV-ctrl or LV-Notch1-shRNA, for 7 days, followed by MOG 35-55 immunization for 23 days ( n = 10 mice per group). a The clinical scores of EAE mice with LV-ctrl and LV-Notch1-shRNA . b The expressions of IL-9, GFAP, NICD, and p-STAT3 in the spinal cords were detected using western blot assay. c , d The changes of IL-6, TNF-α, and IP-10 in the spinal cords and peripheral blood of the LV-ctrl and LV-Notch1-shRNA mice were evaluated by real-time PCR and CBA assay, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. PBS group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus LV-ctrl group (one-way ANOVA, the two-tailed Student’s t test). Results are represented as mean ± SEM. e The infiltration of inflammatory cells in spinal cords was investigated using hematoxylin and eosin (H&E) staining (scale bars, 50 μm). f The medullary sheath damages from spinal cords were observed via luxol fast blue (LFB) staining (scale bars, 50 μm). Boxed areas in the upper rows are presented enlarged underneath

Article Snippet: Antibodies used in this study were as follows: anti-IL-9 antibody (Rabbit, ab203386, Abcam), anti-GFAP antibody (Rabbit, ab7260, Abcam), anti-GFAP (Mouse, ab4648, Abcam), anti-cleaved Notch1 antibody (NICD, Rabbit, 4147, Cell Signaling Technology), anti-Notch 1 antibody (Goat, sc-6014, SANTA CRUZ), anti-p-STAT3 (Rabbit, Tyr705, BS4181, Bioworld Technology), anti-STAT3 (Rabbit, AP0365, Bioworld Technology), NF-κB p65 (Rabbit, 8242, Cell Signaling Technology), anti- CBP/P300 antibody (Rabbit, 4771s, Cell Signaling Technology), anti-RNA Pol II antibody (Mouse, 17-620, Sigma Aldrich), and anti-H3K27ac antibody (Rabbit, ab177178, Abcam).

Techniques: Knockdown, Recombinant, shRNA, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test, Staining

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Oligonucleotides sequences used for quantifying miR-141 and mRNA of indicated genes

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques:

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: The correlation between miR-141 level and the expression profile of MxA and STAT3 in infected A549 cells. (A) Quantification of steady-state miR-141 in infected A549 cells with MOI of 0.5 and transfected with either pre-miR-141 or miR-141 inhibitor compared with noninfected cells (control) using qRT-PCR. (B) Relative gene expression of MxA and STAT3 in infected A549 cells transfected with either specific inhibitor against miR-141 or pre- miR-141 compared with control-transfected cells using qRT-PCR. Error bars indicate the STD of three independent experiments. Student two-tailed t -test used for statistical analysis, (*) indicates P-values ≤ 0.05, and (**) indicates P ≤ 0.01. (C) Flow cytometric assay quantifies the kinetic proteins expression profile of MxA (in blue dots) and STAT3 (in red dots) in infected and transfected A549 cells compared with control cells. (D) Western blot analysis reveals the protein expression level of MxA and STAT3 in infected and transfected cells compared to control cells, β-actin expression profile severed as an internal control

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Expressing, Infection, Transfection, Control, Quantitative RT-PCR, Gene Expression, Two Tailed Test, Flow Cytometry, Western Blot

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Quantification analysis of miR-141, MxA, and STAT3 in transfected and infected A549 cells

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Transfection, Infection, Expressing, Control