Journal: Hepatology Communications

Article Title: Fatty acid β-oxidation enhances immune regulatory function of double-negative T cells through pSTAT4–OX40 signaling pathway

doi: 10.1097/HC9.0000000000000783

Figure Lengend Snippet: OX40 expression is regulated by STAT signaling. (A) Bioinformatic prediction of STAT4-binding sites in the promoter region of OX40 (−1500 bp/+100 bp region). (B) Relative chemiluminescence signal of the STAT family with or without the binding of the OX40 promoter. (C) Flow cytometry of phosphorylated STAT4 expression level of DNT cells with the supplement of FAO inhibitor Eto. (D) Western blot of total and phosphorylated STAT4 expression level of DNT cells with the supplement of Eto, and statistical analysis of the relative p-STAT4/total STAT4 ratio. (E) Flow cytometry of OX40 expression level of DNT cells with the supplement of STAT4 inhibitor, LSF. (F) Flow cytometry of Annexin V detection and Bcl-2 expression level of DNT cells with the supplement of LSF. (G) Flow cytometry of the Ki67 expression level of DNT cells with the supplement of LSF. (H) Flow cytometry of cytotoxicity-related molecules PRF1 and GzmB in DNT cells with the supplement of LSF. (I) Flow cytometry of activation-related molecules NKG2D and NKG2A in DNT cells with the supplement of LSF. * p <0.05. Abbreviations: DNT, double-negative T; Eto, Etomoxir; FAO, fatty acid β-oxidation; GzmB, Granzyme B; LSF, Lisofylline.

Article Snippet: The fatty acids palmitic acid (PA, Sigma-Aldrich) and oleic acid (OA, Sigma-Aldrich), the FAO inhibitor etomoxir (Eto, Sigma-Aldrich), and the STAT4 inhibitor (R)-lisofylline (MCE) were added to the culture medium.

Techniques: Expressing, Binding Assay, Flow Cytometry, Western Blot, Activation Assay

Journal: Hepatology Communications

Article Title: Fatty acid β-oxidation enhances immune regulatory function of double-negative T cells through pSTAT4–OX40 signaling pathway

doi: 10.1097/HC9.0000000000000783

Figure Lengend Snippet: FAO contributes to the therapeutic effect of DNT cells on ConA-induced autoimmune liver models. (A) TUNEL staining schematic diagrams of control, ConA, DNT treatment, and DNT-Eto treatment groups, and a statistical chart of relative TUNEL fluorescence. (B) H&E staining schematic diagrams of control, ConA, DNT treatment, and DNT-Eto treatment groups. (C) Statistical chart of liver necrosis area of control, ConA, DNT treatment, and DNT-Eto treatment group. (D) Serum ALT and AST levels of control, ConA, DNT treatment, and DNT-Eto treatment group. (E) Flow cytometry analysis of intrahepatic CD4 + T, CD8 + T, and NKT cells in different treatment groups. (F) Flow cytometry of Annexin V detection of DNT cells in the DNT treatment and DNT-Eto treatment groups. (G) Flow cytometry of Ki67 levels of DNT cells in the DNT treatment and DNT-Eto treatment groups. (H) Flow cytometry of Bcl-2, GzmB, and NKG2D levels of DNT cells in the DNT treatment and DNT-Eto treatment groups. (I) Flow cytometry of OX40 expression levels of DNT cells in the DNT treatment and DNT-Eto treatment groups. (J) Flow cytometry of relative p-STAT4 expression levels of DNT cells in the DNT treatment and DNT-Eto treatment groups. * p <0.05. Abbreviations: ConA, concanavalin A; DNT, double-negative T; Eto, etomoxir; FAO, fatty acid β-oxidation; GzmB, Granzyme B; H&E, hematoxylin and eosin; NKT, natural killer T.

Article Snippet: The fatty acids palmitic acid (PA, Sigma-Aldrich) and oleic acid (OA, Sigma-Aldrich), the FAO inhibitor etomoxir (Eto, Sigma-Aldrich), and the STAT4 inhibitor (R)-lisofylline (MCE) were added to the culture medium.

Techniques: TUNEL Assay, Staining, Control, Fluorescence, Flow Cytometry, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Genetic and Functional Characterization of STAT4 in Rheumatoid Arthritis Patients with Distinct Disease Activity

doi: 10.3390/ijms262010011

Figure Lengend Snippet: Comparison of IL-12, IL-23, IFN-γ, and anti-CPP levels across rs7574865 genotypes of the STAT4 gene. Each bar represents a genotype (GG, GT, TT). Group 1 corresponds to RA patients in remission/low disease activity, and Group 2 corresponds to patients with moderate/high disease activity. ( A ) Serum IL-12 levels in both activity groups, showing no significant differences among genotypes. ( B ) Anti-CCP antibody concentrations in Group 2 were higher in TT and GT genotypes compared with GG, whereas in Group 1, higher values were observed in the TT genotype; however, differences were not statistically significant. ( C ) IFN-γ concentrations did not differ significantly between genotypes in either group. ( D ) IL-23 levels were similar across genotypes and did not reach statistical significance. Statistical comparisons were performed using the Kruskal–Wallis nonparametric test with Dunn’s post hoc comparison. Numbers above bars indicate the number of patients per genotype in each group.

Article Snippet: Genomic DNA from 63 patients was genotyped by allelic discrimination assays using predesigned TaqMan ® probes for the rs7574865 (ID: C_29882391_10) and rs11889341 (ID: C_26419582_10) variants (Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Genetic and Functional Characterization of STAT4 in Rheumatoid Arthritis Patients with Distinct Disease Activity

doi: 10.3390/ijms262010011

Figure Lengend Snippet: Comparison of IL-12, IFN-γ, IL-23, and PBMCs counts across STAT4 haplotype combinations. Each bar represents a haplotype combination (GC/GC, GC/TT, TT/TT). Group 1 corresponds to RA patients in remission/low disease activity, and Group 2 corresponds to patients with moderate/high disease activity. ( A ) Serum IL-12 levels in both groups. Patients carrying the GC/GC haplotype combination in Group 1 exhibited significantly higher IL-12 concentrations compared with those with GC/TT or TT/TT ( p < 0.01). No significant differences were observed among haplotype combinations in Group 2. ( B ) IFN-γ concentrations did not differ significantly between haplotype combinations in either group. ( C ) IL-23 levels were similar across haplotypes and did not reach statistical significance. ( D ) PBMC counts were significantly higher for the GC/GC haplotype compared with GC/TT in Group 1 ( p < 0.01), whereas no differences were detected among haplotypes in Group 2. Statistical comparisons were performed using the Kruskal–Wallis nonparametric test with Dunn’s post hoc comparison. Numbers above bars indicate the number of patients per haplotype in each group. ** Significance value < 0.01.

Article Snippet: Genomic DNA from 63 patients was genotyped by allelic discrimination assays using predesigned TaqMan ® probes for the rs7574865 (ID: C_29882391_10) and rs11889341 (ID: C_26419582_10) variants (Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Genetic and Functional Characterization of STAT4 in Rheumatoid Arthritis Patients with Distinct Disease Activity

doi: 10.3390/ijms262010011

Figure Lengend Snippet: Comparison of diagnostic parameters across STAT4 haplotype combinations. Each bar represents a haplotype combination (GC/GC, GC/TT, TT/TT). ( A ) Serum anti-CCP levels were significantly higher in the GC/GC haplotype combination within the remission-low activity group compared to GC/TT ( p < 0.01). No significant differences were observed among haplotype combinations in the moderate-high activity group. ( B ) ESR was significantly higher in the GC/TT haplotype combination compared with TT/TT in the moderate-high activity group ( p < 0.05). ( C ) CRP and ( D ) RF showed no significant differences among haplotype combinations. Statistical comparisons were performed using the Kruskal–Wallis nonparametric test with Dunn’s post hoc comparison. Numbers above bars indicate the number of patients per haplotype in each group. ** Significance value < 0.01; * Significance value < 0.05.

Article Snippet: Genomic DNA from 63 patients was genotyped by allelic discrimination assays using predesigned TaqMan ® probes for the rs7574865 (ID: C_29882391_10) and rs11889341 (ID: C_26419582_10) variants (Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Diagnostic Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Genetic and Functional Characterization of STAT4 in Rheumatoid Arthritis Patients with Distinct Disease Activity

doi: 10.3390/ijms262010011

Figure Lengend Snippet: Comparison of STAT4 protein and gene expression across STAT4 haplotype combinations. Each bar represents a haplotype combination (GC/GC, GC/TT, TT/TT). ( A ) Expression levels of phosphorylated STAT4 (pSTAT4) and ( B ) STAT4 mRNA expression showed no significant differences among haplotype combinations in either the remission-low activity group or the moderate-high activity group. Basal pSTAT4 measurements were performed in non-stimulated PBMCs from patients receiving conventional DMARD therapy, which may influence basal phosphorylation and should be considered when interpreting these results. Statistical comparisons were performed using the Kruskal–Wallis nonparametric test with Dunn’s post hoc comparison. Numbers above bars indicate the number of patients per haplotype in each group.

Article Snippet: Genomic DNA from 63 patients was genotyped by allelic discrimination assays using predesigned TaqMan ® probes for the rs7574865 (ID: C_29882391_10) and rs11889341 (ID: C_26419582_10) variants (Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Gene Expression, Expressing, Activity Assay, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Genetic and Functional Characterization of STAT4 in Rheumatoid Arthritis Patients with Distinct Disease Activity

doi: 10.3390/ijms262010011

Figure Lengend Snippet: Comparison of inflammatory biomarkers according to STAT4 mRNA expression levels. ( A ) Serum IL-12 levels showed no significant differences in the STAT4 underexpression group ( p > 0.05). ( B , C ) Anti-CCP antibody and IFN-γ levels were comparable in the underexpression group and did not reach statistical significance. ( D ) IL-23 levels showed no significant differences in the underexpression group. Light gray bars represent patients with STAT4 underexpression, and dark gray bars represent patients with STAT4 overexpression. Results for the overexpression group across all analyzed markers are presented descriptively and should be interpreted with caution due to the small sample size. Numbers above bars indicate the number of patients per haplotype in each group.

Article Snippet: Genomic DNA from 63 patients was genotyped by allelic discrimination assays using predesigned TaqMan ® probes for the rs7574865 (ID: C_29882391_10) and rs11889341 (ID: C_26419582_10) variants (Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Expressing, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Genetic and Functional Characterization of STAT4 in Rheumatoid Arthritis Patients with Distinct Disease Activity

doi: 10.3390/ijms262010011

Figure Lengend Snippet: Comparison of serum IL-12 and IL-23 levels according to STAT4 haplotypes and mRNA expression levels in rheumatoid arthritis patients with different disease activity. ( A , B ) In the remission–low activity group (Group 1), patients with STAT4 underexpression and the GC/GC haplotype had significantly higher IL-12 levels compared with those carrying GC/TT ( p < 0.05) or TT/TT ( p < 0.01). In contrast, in the moderate–high activity group (Group 2), results for patients with STAT4 overexpression are shown descriptively only and were not included in the statistical analyses. ( C , D ) For IL-23, patients with STAT4 underexpression did not show significant differences between haplotypes ( p = 0.07). Results from the STAT4 overexpression subgroup in both activity groups are presented descriptively only. Numbers above bars indicate the number of patients per haplotype in each group. ** Significance value < 0.01; * Significance value < 0.05.

Article Snippet: Genomic DNA from 63 patients was genotyped by allelic discrimination assays using predesigned TaqMan ® probes for the rs7574865 (ID: C_29882391_10) and rs11889341 (ID: C_26419582_10) variants (Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Expressing, Activity Assay, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Genetic and Functional Characterization of STAT4 in Rheumatoid Arthritis Patients with Distinct Disease Activity

doi: 10.3390/ijms262010011

Figure Lengend Snippet: Comparison of IL-12, IL-23, IFN-γ, and anti-CPP levels across rs7574865 genotypes of the STAT4 gene. Each bar represents a genotype (GG, GT, TT). Group 1 corresponds to RA patients in remission/low disease activity, and Group 2 corresponds to patients with moderate/high disease activity. ( A ) Serum IL-12 levels in both activity groups, showing no significant differences among genotypes. ( B ) Anti-CCP antibody concentrations in Group 2 were higher in TT and GT genotypes compared with GG, whereas in Group 1, higher values were observed in the TT genotype; however, differences were not statistically significant. ( C ) IFN-γ concentrations did not differ significantly between genotypes in either group. ( D ) IL-23 levels were similar across genotypes and did not reach statistical significance. Statistical comparisons were performed using the Kruskal–Wallis nonparametric test with Dunn’s post hoc comparison. Numbers above bars indicate the number of patients per genotype in each group.

Article Snippet: Genomic DNA from 63 patients was genotyped by allelic discrimination assays using predesigned TaqMan ® probes for the rs7574865 (ID: C_29882391_10) and rs11889341 (ID: C_26419582_10) variants (Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Genetic and Functional Characterization of STAT4 in Rheumatoid Arthritis Patients with Distinct Disease Activity

doi: 10.3390/ijms262010011

Figure Lengend Snippet: Comparison of IL-12, IFN-γ, IL-23, and PBMCs counts across STAT4 haplotype combinations. Each bar represents a haplotype combination (GC/GC, GC/TT, TT/TT). Group 1 corresponds to RA patients in remission/low disease activity, and Group 2 corresponds to patients with moderate/high disease activity. ( A ) Serum IL-12 levels in both groups. Patients carrying the GC/GC haplotype combination in Group 1 exhibited significantly higher IL-12 concentrations compared with those with GC/TT or TT/TT ( p < 0.01). No significant differences were observed among haplotype combinations in Group 2. ( B ) IFN-γ concentrations did not differ significantly between haplotype combinations in either group. ( C ) IL-23 levels were similar across haplotypes and did not reach statistical significance. ( D ) PBMC counts were significantly higher for the GC/GC haplotype compared with GC/TT in Group 1 ( p < 0.01), whereas no differences were detected among haplotypes in Group 2. Statistical comparisons were performed using the Kruskal–Wallis nonparametric test with Dunn’s post hoc comparison. Numbers above bars indicate the number of patients per haplotype in each group. ** Significance value < 0.01.

Article Snippet: Genomic DNA from 63 patients was genotyped by allelic discrimination assays using predesigned TaqMan ® probes for the rs7574865 (ID: C_29882391_10) and rs11889341 (ID: C_26419582_10) variants (Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Genetic and Functional Characterization of STAT4 in Rheumatoid Arthritis Patients with Distinct Disease Activity

doi: 10.3390/ijms262010011

Figure Lengend Snippet: Comparison of diagnostic parameters across STAT4 haplotype combinations. Each bar represents a haplotype combination (GC/GC, GC/TT, TT/TT). ( A ) Serum anti-CCP levels were significantly higher in the GC/GC haplotype combination within the remission-low activity group compared to GC/TT ( p < 0.01). No significant differences were observed among haplotype combinations in the moderate-high activity group. ( B ) ESR was significantly higher in the GC/TT haplotype combination compared with TT/TT in the moderate-high activity group ( p < 0.05). ( C ) CRP and ( D ) RF showed no significant differences among haplotype combinations. Statistical comparisons were performed using the Kruskal–Wallis nonparametric test with Dunn’s post hoc comparison. Numbers above bars indicate the number of patients per haplotype in each group. ** Significance value < 0.01; * Significance value < 0.05.

Article Snippet: Genomic DNA from 63 patients was genotyped by allelic discrimination assays using predesigned TaqMan ® probes for the rs7574865 (ID: C_29882391_10) and rs11889341 (ID: C_26419582_10) variants (Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Diagnostic Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Genetic and Functional Characterization of STAT4 in Rheumatoid Arthritis Patients with Distinct Disease Activity

doi: 10.3390/ijms262010011

Figure Lengend Snippet: Comparison of STAT4 protein and gene expression across STAT4 haplotype combinations. Each bar represents a haplotype combination (GC/GC, GC/TT, TT/TT). ( A ) Expression levels of phosphorylated STAT4 (pSTAT4) and ( B ) STAT4 mRNA expression showed no significant differences among haplotype combinations in either the remission-low activity group or the moderate-high activity group. Basal pSTAT4 measurements were performed in non-stimulated PBMCs from patients receiving conventional DMARD therapy, which may influence basal phosphorylation and should be considered when interpreting these results. Statistical comparisons were performed using the Kruskal–Wallis nonparametric test with Dunn’s post hoc comparison. Numbers above bars indicate the number of patients per haplotype in each group.

Article Snippet: Genomic DNA from 63 patients was genotyped by allelic discrimination assays using predesigned TaqMan ® probes for the rs7574865 (ID: C_29882391_10) and rs11889341 (ID: C_26419582_10) variants (Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Gene Expression, Expressing, Activity Assay, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Genetic and Functional Characterization of STAT4 in Rheumatoid Arthritis Patients with Distinct Disease Activity

doi: 10.3390/ijms262010011

Figure Lengend Snippet: Comparison of inflammatory biomarkers according to STAT4 mRNA expression levels. ( A ) Serum IL-12 levels showed no significant differences in the STAT4 underexpression group ( p > 0.05). ( B , C ) Anti-CCP antibody and IFN-γ levels were comparable in the underexpression group and did not reach statistical significance. ( D ) IL-23 levels showed no significant differences in the underexpression group. Light gray bars represent patients with STAT4 underexpression, and dark gray bars represent patients with STAT4 overexpression. Results for the overexpression group across all analyzed markers are presented descriptively and should be interpreted with caution due to the small sample size. Numbers above bars indicate the number of patients per haplotype in each group.

Article Snippet: Genomic DNA from 63 patients was genotyped by allelic discrimination assays using predesigned TaqMan ® probes for the rs7574865 (ID: C_29882391_10) and rs11889341 (ID: C_26419582_10) variants (Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Expressing, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Genetic and Functional Characterization of STAT4 in Rheumatoid Arthritis Patients with Distinct Disease Activity

doi: 10.3390/ijms262010011

Figure Lengend Snippet: Comparison of serum IL-12 and IL-23 levels according to STAT4 haplotypes and mRNA expression levels in rheumatoid arthritis patients with different disease activity. ( A , B ) In the remission–low activity group (Group 1), patients with STAT4 underexpression and the GC/GC haplotype had significantly higher IL-12 levels compared with those carrying GC/TT ( p < 0.05) or TT/TT ( p < 0.01). In contrast, in the moderate–high activity group (Group 2), results for patients with STAT4 overexpression are shown descriptively only and were not included in the statistical analyses. ( C , D ) For IL-23, patients with STAT4 underexpression did not show significant differences between haplotypes ( p = 0.07). Results from the STAT4 overexpression subgroup in both activity groups are presented descriptively only. Numbers above bars indicate the number of patients per haplotype in each group. ** Significance value < 0.01; * Significance value < 0.05.

Article Snippet: Genomic DNA from 63 patients was genotyped by allelic discrimination assays using predesigned TaqMan ® probes for the rs7574865 (ID: C_29882391_10) and rs11889341 (ID: C_26419582_10) variants (Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Expressing, Activity Assay, Over Expression