Fig 2: Knockout of TRIM22 inhibits the expression of proteins in the RIG-I/NF-κB/CCAR1 pathway(A) Expression of RIG-I/NF-κB/CCAR1 pathway proteins between different groups. (B) The binding ability of RIG-I and MAVS in different modified U251MG cells was detected by coIP. (C) Binding sites for the transcription factor NF-κB and IRF-3 to CCAR1 promoter. (D) Dual-luciferase reporter assay of NF-κB and IRF-3 for CCAR1-promoter. FL/RL, Firefly luciferase/Renilla luciferase. Renilla activity was used to normalize luciferase reporter activity. The promoterless firefly-luciferase vector pGL4.15 served as the negative control (NC). (E) H&E staining of mouse brain tissue sections. T, tumor; N, normal tissue. (F) Immunohistochemical staining of RIG-I/NF-κB/CCAR1 pathway in mouse brain tissue sections. T, tumor; N, normal tissue. (G) Clonogenic situation after knockdown or overexpression of RIG-I, NF-κB, and CCAR1; control on the top panel: control sequence of siRNA, invalid sequence; control of bottom panel: control sequence of overexpression plasmid, empty vector. GAPDH is used as a housekeeping protein to prove the equal loading in each lane. ∗p < 0.05 and ∗∗∗p < 0.001. Each experiment was repeated three times.

Fig 3: Expression and survival analysis of TRIM22 in clinical patients(A) H&E staining of different grades of glioma (10×). (B) Expression of RIG-I/NF-κB/CCAR1 pathway mRNA in HGGs and LGGs. (C) Survival analysis of GBM and LGGs. (D) Survival analysis of LGGs and GBM with different TRIM22 expression. (E) Co-expression of TRIM22, NT5C2, and RIG-I in different grades of glioma (20×). (F) Expression of TRIM22, NT5C2, RIG-I, NF-κB, CCAR1, Ki-67, IDH-1, and MGMT in grade II (40×) and IV gliomas (40×). (G) The survival rate of patients with different TRIM22 expression levels in different treatment groups was analyzed. Ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Each experiment was repeated three times.

Fig 4: Knockout of MAP4K1 potentiates RLR-mediated signaling. (A, B) MAP4K1-knockout THP1 cells were generated by CRISPR-Cas9 technology. MAP4K1-knockout THP1 cells were analyzed by immunoblotting (A) and sequencing analysis (B). (C) MAP4K1 knockout enhanced the dimerization of IRF3. MAP4K1 knockout cells (2 × 106) were uninfected or infected by SeV for 8 h and collected for dimerization assay by native PAGE. (D, E) Real-time qPCR assays of MAP4K1-knockout cells transfected with poly I:C (1 μg) (D) or infected with SeV for 8 h (E). Error bars indicate SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significant difference.

Fig 5: Identification of the type of ubiquitination for TBK1/IKKε. (A to D) 293T cells (∼4 × 106) were transfected with Flag-tagged TBK1 or IKKε (8 μg) and Myc-MAP4K1 (8 μg) together with HA-tagged K48 (A, B) or K63 ubiquitin (8 μg) (C, D). After transfection for 12 h, the cells were treated with SeV or not for 10 h and MG132 for 8 h. Ubiquitination levels were detected by the method described above and immunoblotting analysis with indicated antibodies. (E, F) 293T cells were transfected with Myc-tagged TBK1 (E) or IKKε (F), HA-tagged K48 ubiquitin, and Flag-MAP4K1 together with DTX4 shRNA or control. The cells were treated with MG132 for 8 h. Coimmunoprecipitation and immunoblotting analyses of extracts were performed with indicated antibodies.