Journal: Scientific Reports

Article Title: Anti-interleukin-6 receptor antibody treatment ameliorates postoperative adhesion formation

doi: 10.1038/s41598-019-54175-1

Figure Lengend Snippet: Proinflammatory responses prior to pro-fibrotic alterations. ( a,c ) Cecum lesions were sampled from each experimental group (3–5 mice/group) at the indicated time points post operation, followed by qRT-PCR assessment of expression of proinflammatory cytokine and pro-fibrotic molecule genes ( a ) and by immunostaining of phosphorylated transcription factors with ImageJ analysis data ( c,d ) Ifng , interferon γ; Il6 , interleukin-6; Tnf , tumor necrosis factor-α; Pai1 , plasminogen activator inhibitor type 1; Col1a1 , collagen 1α1; Tgfb1 , tumor growth factor- β1. ( b ) Peritoneal fluid and serum were sampled from human patients at the indicated time points following the beginning of laparotomy, and IL-6 levels were measured using ELISA. Representative photos are shown. Data at 0 day or hour postoperation indicated those in untreated control mice. Data are shown as mean ± SD. * p < 0.05 (Dunnett’s test). Daggers indicate serosa; open brackets indicate adhesion lesions. All experiments were independently repeated twice.

Article Snippet: We measured the abundance of IL-6, TNF-α, TGF-β1 and sIL-6Rα in sera of mice with mouse ELISA kits of IL-6 (M6000B, R&D Systems), TNF-α (MTA00B, R&D Systems), TGF-β1 (MB100B, R&D Systems), and sIL-6Rα (DY1830, R&D Systems), respectively.

Techniques: Quantitative RT-PCR, Expressing, Immunostaining, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK is highly expressed in and required for Th17 cells. (A) Naïve CD4 T cells were cultured under each subset differentiation conditions for 3 days. The transcript level of Fak was measured by RT-qPCR (left) and the protein level of FAK was measured by immunoblot analysis (right). (B, C) Naïve CD4 T cells from Fak fl/fl mice were introduced with a control empty vector (control) or a CRE recombinase-expressing vector (RV-Cre) to induce Fak deletion and cultured under Th17-polarizing conditions for 3 days (B) or various subset-polarizing conditions (C) for 3 days. (B) GFP+ cells were sorted. The transcript level of Fak was measured by RT-qPCR (left) and the protein level of FAK was measured by immunoblot analysis (right). (C) The expression of GFP, IFN-γ, IL-4, IL-17A, and FOXP3 was analyzed by flow cytometry (top). The statistical analysis was performed on pooled data from five independent experiments (bottom). Error bars represent the standard deviation. The significance of differences between groups was determined by one-way ANOVA (A) and Student t test (B, C) . ***P < 0.001; ****P < 0.0001, n.s., not significant.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Control, Plasmid Preparation, Expressing, Flow Cytometry, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK affects Th17 cell differentiation program. (A, B) Naïve CD4 T cells from Fak fl/fl mice were cultured and sorted as Figure 1B . (A) IL-17A+ and FOXP3+ cells were measured by flow cytometry. (B) Transcript levels of Il17a , Rorc , Il23r , and Foxp3 were measured by RT-qPCR. (C, D) Naïve CD4 T cells were introduced with either the control vector (MSCV-LMP) or Fak shRNA vectors (#1, #2, and #3) and cultured under Th17-polarizing conditions for 3 days. (C) The transcript level of Fak was measured by RT-qPCR (left) and protein level of FAK was measured by immunoblot analysis (right). (D) IL-17A+ cells among the vector-transduced cells (GFP+) were measured by flow cytometry (left). GFP+ cells were sorted and the transcript level of Il17a was measured by RT-qPCR (right). All of RT-qPCR data were normalized to Gapdh . (E–G) Naïve CD4 T cells were transduced with control or RV-Cre and cultured under Th17-polarizing conditions for 3 days. GFP+ cells were sorted and subjected to RNA-seq analysis. (E) Scatter plot of RNA-seq data. (F) Gene ontology analysis of differentially expressed genes (DEGs) from control and RV-Cre-transduced Th17 cells. (G) Heatmap of immune/inflammatory response-related genes among the DEGs from control and RV-Cre-transduced Th17 cells. All of RT-qPCR data were normalized to Gapdh . Data in (A–D) are pooled from three independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Differentiation, Cell Culture, Flow Cytometry, Quantitative RT-PCR, Control, Plasmid Preparation, shRNA, Western Blot, Transduction, RNA Sequencing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK deficiency ameliorates the severity of EAE. (A, B) Naïve CD4 T cells from WT and Fak fl/fl Rorc cre mice were cultured under polarizing conditions toward each different subset for 3 days. Transcript levels of Ifng for Th1 cells, Il4 for Th2 cells, Il17a for Th17 cells, and Foxp3 for Treg cells were measured by RT-qPCR (A) and the percentage of IL-17A+ cells under Th17-polarizing conditions was measured by flow cytometry (B) . (C) EAE was induced in control (WT, n = 10) and Fak fl/fl Rorc cre (n = 10) mice as described in the Materials and Methods section. The symptoms of EAE were monitored every day and clinical scores were evaluated after EAE induction. (D, E) Histopathological analysis of lumbar spinal cords from control and Fak fl/fl Rorc cre mice at the peak of the disease. (D) H&E stained sections of spinal cords. Arrows indicate the inflammatory foci. (E) Immunohistochemical staining of myelin basic protein (MBP). Arrows indicate the more preserved myelin in Fak fl/fl Rorc cre mice compared with control mice. (F) IL-17A+ and IFNγ+ cells (left) and FOXP3+ cells (right) among CNS-infiltrating CD4 T cells were measured by flow cytometry. (G–J) The percentage and absolute number of CNS-infiltrating mononuclear cells were measured. (G) CD4+ cells, (H) IL-17A+ cells, (I) IFNγ+ cells, and (J) FOXP3+ cells. (K) Transcript levels of Il17a , Rorc , Il23r , Ifng , and Foxp3 in CNS-infiltrating mononuclear cells were measured by RT-qPCR. Data were normalized to Gapdh . Data in (F–K) are pooled from ten independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, n.s., not significant.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Culture, Quantitative RT-PCR, Flow Cytometry, Control, Staining, Immunohistochemical staining, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK regulates the STAT3 signaling pathway in Th17 cells. (A) Naïve CD4 T cells were cultured under various differentiation conditions for the indicated time periods and the transcript levels of Fak , Il17a , Rorc , and Il17f were measured by RT-qPCR. (B) Naïve CD4 T cells were introduced with control vector or Rorc -expressing vector ( Rorc O/X) and cultured in Th17-polarizing conditions for 3 days. Transcript levels of Rorc , Il17a , and Fak were measured by RT-qPCR. (C) Naïve CD4 T cells were cultured under Th17-polarizing conditions with dose-dependent treatment of GSK805 for 3 days. Transcript levels of Il17a and Fak were measured by RT-qPCR. (D, E) Naïve CD4 T cells from Fak fl/fl mice were cultured as described in Figure 1B and GFP+ cells were sorted. (D) Each protein level was measured by immunoblot analysis (left) and the ratios of pSTAT3/STAT3 and pSTAT5/STAT5 were calculated by densitometry (right). (E) Transcript level of Il2 was measured by RT-qPCR (left) and the protein level of IL-2 from the supernatants was measured by ELISA (right). All of RT-qPCR data were normalized to Gapdh . Data in (A, B, D, E) were pooled from three independent experiments, and data in (C) from five. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. **P < 0.01; ***P < 0.001; ****P < 0.0001, n.s., not significant.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Culture, Quantitative RT-PCR, Control, Plasmid Preparation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: FAK regulates Th17 cell differentiation through the NF-κB pathway. (A) Naïve CD4 T cells from Fak fl/fl mice were transduced with control vector or RV-Cre and cultured under Th17-polarizing conditions for 3 days. GFP+ cells were sorted and rested in normal media for 2 days and serum-free medium for an additional 8 hours. The cells were restimulated with anti-CD3/CD28 antibodies for the indicated time periods, and nuclear/cytoplasmic extracts were prepared. Each protein level was measured by immunoblot analysis (right). The ratios of IκB/β-Actin, pIκB/β-Actin, and nuclear RelA/cytoplasmic RelA were calculated by densitometry (right). (B, C) Naïve CD4 T cells from Fak fl/fl mice were transduced with RV-Cre and cultured under Th17-polarizing conditions for 3 days (B) and 16 hours (C) and GFP+ cells were sorted. (B) Transcript level of Socs3 was measured by RT-qPCR. (C) Relative RelA binding to each indicated locus was measured through ChIP assay. Nuclear extracts from cultured cells were reacted with an anti-RelA antibody, and precipitated DNA fragments were measured by qPCR. Isotype-matching IgG was used as a negative control. (D–F) Il17a promoter activity was measured through luciferase assay. (D) EL4 cells were transfected with pGL3- Il17a promoter vector and control or Fak siRNA, and the cells were rested for 20 hours. After transfection, the cells were divided into non-stimulated or stimulated groups, with the stimulated groups received 4 hour stimulation with PMA/ionomycin. (E) EL4 cells were transfected with the pGL3- Il17a promoter vector and rested for 20 hours with or without PDTC treatment (1 μM). (F) EL4 cells were transfected as described in (D) and rested for 20 hours with 1 μM PDTC treatment. (G–I) Naïve CD4 T cells from Rela fl/fl mice were introduced with a control empty vector (WT) or a Cre recombinase expressing vector (p65 KO) to induce Rela deletion and cultured under Th17-polarizing conditions for 3 days. (G) IL-17A+ and FOXP3+ cells were measured by flow cytometry. (H) GFP+ cells were sorted and transcript level of Rela , Il17a , Rorc , and Il23r were measured by RT-qPCR. (I) Naïve CD4 T cells from Rela fl/fl mice were cultured as described in (G) and additionally treated with vehicle (control) or FAK inhibitor (PND1186, 1 μM). IL-17A+ and FOXP3+ cells were measured by flow cytometry. RT-qPCR data in (B, H) were normalized to Gapdh . Data in (A–I) are pooled from three independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: Cell Differentiation, Transduction, Control, Plasmid Preparation, Cell Culture, Western Blot, Quantitative RT-PCR, Binding Assay, Negative Control, Activity Assay, Luciferase, Transfection, Expressing, Flow Cytometry, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Focal adhesion kinase plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling

doi: 10.3389/fimmu.2025.1596802

Figure Lengend Snippet: A FAK inhibitor blocks differentiation of Th17 cells in vitro. (A–E) Naïve CD4 T cells were cultured under Th17- or Treg-polarizing conditions with dose-dependent treatment of PND1186 for 3 days. IL-17A+ and FOXP3+ cells were measured by flow cytometry (A) and transcript levels of Il17a and Foxp3 were measured by RT-qPCR (C) in Th17 cells. FOXP3+ cells were measured by flow cytometry (B) and transcript level of Foxp3 was measured by RT-qPCR (D) in Treg cells. (E–G) Naïve CD4 T cells were cultured under Th17-polarizing conditions with vehicle (control) or PND1186 (1 μM) treatment for 3 days (E, G) and the indicated time periods (F) . (E) pSTAT3 and pSTAT5 levels were measured by flow cytometry. (F) Nuclear or cytoplasmic extracts were prepared from cultured cells, and each protein level was measured by immunoblot analysis (right). The ratios of IκB/β-Actin, pIκB/β-Actin, and nuclear RelA/cytoplasmic RelA were calculated by densitometry (right). (G) Relative RelA binding to each indicated locus was measured through ChIP assay. Nuclear extracts from cultured cells were incubated with an anti-RelA antibody and the precipitated DNA fragments were measured by qPCR. An isotype-matching IgG was used as a negative control. RT-qPCR data in (C, D) were normalized to Gapdh . Data in (A–G) are pooled from three independent experiments. Error bars represent the standard deviation. The significance of differences between groups was determined by Student t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Th1: mouse recombinant IL-2 (1 ng/ml, eBioscience), mouse recombinant IL-12 p70 (3.5 ng/ml, eBioscience), and anti-mouse IL-4 (5 μg/ml); Th2: mouse recombinant IL-2 (1 ng/ml), mouse recombinant IL-4 (5 ng/ml, R&D systems), and anti-mouse IFNγ (5 μg/ml); Th17: mouse recombinant IL-6 (50 ng/ml, R&D systems), human recombinant TGFβ1 (1 ng/ml, R&D systems), mouse recombinant TNFα (1 ng/ml, eBioscience), mouse recombinant IL-1β (2 ng/ml, Gibco), anti-mouse IFNγ (5 μg/ml), and anti-mouse IL-4 (5 μg/ml); Tregs: mouse recombinant IL-2 (1 ng/ml), human recombinant TGFβ1 (5 ng/ml), anti-mouse IFNγ (10 μg/ml), and anti-mouse IL-4 (10 μg/ml).

Techniques: In Vitro, Cell Culture, Flow Cytometry, Quantitative RT-PCR, Control, Western Blot, Binding Assay, Incubation, Negative Control, Standard Deviation

Journal: OncoTargets and Therapy

Article Title:

Genes Regulated by HPV 16 E6 and High Expression of NFX1-123 in Cervical Cancers

doi: 10.2147/ott.s251926

Figure Lengend Snippet: Figure 3 mRNA expression of genes regulated by HPV 16E6 and NFX1-123 were significantly upregulated in cervical cancer. mRNA expression of 19 genes regulated by 16E6 and NFX1-123 were analyzed using TCGA Target TGEx in normal cervix (n = 10) compared to cervical primary tumors (n = 303). Significance was determined using two-tailed Mann–Whitney test. 14 of 19 genes were significantly upregulated in cervical tumors compared to normal tissue. LOR, CEBPD, LCE2B, FOXA2, and FBN2 were deregulated in cervical cancers, although they did not reach statistical significance. Note the Y-axis scales for individual gene expression datasets were adjusted to demonstrate samples with no detectable gene expression (0 value).

Article Snippet: Protein detection was performed with the following primary antibodies: RAET1G (Mouse RAET1G, 1:100 dilution, Santa Cruz Biotechnology, Cat No. sc-53134, Dallas, TX, USA); Notch1 (Rat Notch1 5B5, which detects both full-length Notch1 at 300kDa and its inactive NTM domain at 120kDa, 1:500 dilution, Cell Signaling Technology, Cat No.3447, Danvers, MA, USA); Transglutaminase 1 (Rabbit TGM1 1:500 dilution, Abcam, Cat No. ab27000, Cambridge, MA, USA); FOXA2 (Mouse HNF-3α/β (E-4) FOXA2, 1:100 dilution, Santa Cruz Biotechnology, Cat No. sc-377033, Dallas, TX, USA), and beta-actin (Mouse beta-actin (AC-15), 1:2000 dilution, Invitrogen, Cat No. AM4302, Waltham, MA USA).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Gene Expression

Journal: OncoTargets and Therapy

Article Title:

Genes Regulated by HPV 16 E6 and High Expression of NFX1-123 in Cervical Cancers

doi: 10.2147/ott.s251926

Figure Lengend Snippet: Figure 5 Protein expression of genes upregulated with overexpressed NFX1-123 and 16E6 in HPV positive cell lines. Western blot analysis of NFX1-123 and 16E6 regulated proteins (A) RAET1G and Notch1 (B) TGM1 and (C) FOXA2 in primary human foreskin keratinocytes (HFK), HPV 16 positive cervical cancer cell lines (SiHa and CaSki), and a HPV 18 positive cervical cancer cell line (HeLa). Beta- actin was used as a loading control. Fold expression of each protein of interest is shown for these cervical cancer cell lines relative to typical expression in HFKs.

Article Snippet: Protein detection was performed with the following primary antibodies: RAET1G (Mouse RAET1G, 1:100 dilution, Santa Cruz Biotechnology, Cat No. sc-53134, Dallas, TX, USA); Notch1 (Rat Notch1 5B5, which detects both full-length Notch1 at 300kDa and its inactive NTM domain at 120kDa, 1:500 dilution, Cell Signaling Technology, Cat No.3447, Danvers, MA, USA); Transglutaminase 1 (Rabbit TGM1 1:500 dilution, Abcam, Cat No. ab27000, Cambridge, MA, USA); FOXA2 (Mouse HNF-3α/β (E-4) FOXA2, 1:100 dilution, Santa Cruz Biotechnology, Cat No. sc-377033, Dallas, TX, USA), and beta-actin (Mouse beta-actin (AC-15), 1:2000 dilution, Invitrogen, Cat No. AM4302, Waltham, MA USA).

Techniques: Expressing, Western Blot, Control