Fig 1: DDX58 regulated the protein stability of STAT1 via the ubiquitin E3 ligase TRIM21 (A). Western blot analysis of the DDX58, P21, and STAT1 proteins and their phosphorylation in DOX- or TMZ-induced senescent LN229 cells transduced with DDX58-targeting siRNAs. (B-C). Western blot analysis of the P21, DDX58, and STAT1 proteins and their phosphorylation in LN229 (B) and U87MG (C) cells overexpressing DDX58. (D). Protein stability assays to assess the effect of DDX58 on the STAT1 protein. DDX58-knockdown senescent LN229 cells were treated with cycloheximide (50 μg/mL) for up to 9 h, and STAT1 and DDX58 expression was tested via western blotting. (E). LN229 cells were transfected with DDX58 siRNA and then treated with MG132 (20 μM) for 5 h. (F). The binding between DDX58 and STAT1 was examined by co-IP and western blotting. (G). LN229 cells were transiently transfected with DDX58 siRNA and then treated with TMZ or DMSO, and the changes in the ubiquitin level of STAT1 in LN229 cells were examined by co-IP and western blotting. All the samples were treated with 20 µM MG132 for 2 h. (H). Regulators involved in the regulation of STAT1 ubiquitination were screened by transient transfection of LN229 cells with the His-STAT1 pcDNA 4.0 plasmid. One sample was treated with 50 μM TMZ for 4 days. All the cells were examined via co-IP and western blotting (I). LN229 cells were transiently transfected with the His-STAT1 plasmid, and changes in the STAT1 binding to TRIM21 in LN229 cells overexpressing DDX58 were examined via co-IP and western blotting. (J). Schematic diagram of the DDX58/RIG-I protein domains. P21 and STAT1 protein expression and phosphorylation by overexpressing the CARD, CTD, and helicase domains of DDX58 in LN229 and 293FT cells. (K). THP-1 macrophages were cocultured with CM from LN229 cells (CON335, DDX58 overexpressing, DDX58 overexpressing plus fludarabine, 50 ng/mL CSF-1) for 48 h. Scale bars: 1.5 mm, 150 μm. (L). Statistical analysis of the data in (K). The samples were analyzed in triplicate with 3 fields per well; ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test. (M). ELISA analysis of CSF1 in LN229 cells overexpressing DDX58 or treated with fludarabine. Comparisons were performed with two-tailed Student’s t tests. *p < 0.01, ***p < 0.001. All the data are presented as the means ± SDs.