(A) Multivariate cox regression analysis of the TRAF family members in TCGA-glioma cohort. (B) Analysis of TRAF3 expression in glioma using TCGA, CGGA, and the Fine brain datasets, respectively. *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Analysis of TRAF3 expression by qRT-PCR in GBM and paired nontumor brain (NTB) tissues (n = 9). *P < 0.05. (D) Analysis of single-cell RNA-Seq data showing the expression of TRAF3 across different cell types in low-grade glioma (LGG) and GBM (SCP1985), respectively. The P values are indicated. Pos, positive. (E) BSP-PCR was used to analyze methylation of the TRAF3 promoter in GBM tissues and paired NTB tissues. The percentage of methylated CpG was statistically analyzed (mean ± SD, n = 11 CpG sites for each tissue). *P < 0.05. (F) MSP analysis of 3 pairs of NTB and GBM tissues. UM, unmethylated; M, methylated. (G) GBM cells were treated with DMSO or 5-azacytidine (5-Aza), and TRAF3 mRNA levels were analyzed by qRT-PCR. GAPDH was used as an internal control. Values were normalized to DMSO (mean ±SD, n = 3 independent experiments). *P < 0.05 and **P < 0.01. (H) TRAF3 protein expression in different grades of gliomas and NTB tissues was analyzed by immunostaining. Representative images are shown. Scale bar: 200 μm. Original magnification, ×200 (insets). Staining for TRAF3 was scored on a scale of 0–12, and the expression scores for TRAF3 in grades III+IV were compared with those for NTB and grades I+II (n = 14 NTB, n = 40 grades I+II, and n = 50 grades III+IV). ***P < 0.001. Statistical analysis was performed using 1-way ANOVA with Tukey’s post hoc test (B and H), paired, 2-tailed Student’s t test (C), χ² test (D), or unpaired, 2-tailed Student’s t test (E and G). G II, grade II; G III, grade III.