Fig 2: UBC9-mediated STAT4 SUMOylation inhibits macrophage activation.(A) TAMs were sorted and analyzed by RNA-Seq from WT and Ubc9–/– PCa-bearing mice. Gene Ontology analysis demonstrated the enriched pathways in TAMs. (B) Heatmap showing genes associated with the JAK/STAT signaling pathway. (C) Immunoprecipitation identified STAT4 SUMOylation in BMDMs treated with BSA or IL-12. (D) SUMOylated STAT4 was obviously detected in BMDMs transduced with Ubc9-overexpressing (Ubc9-OE) adenovirus. (E) SUMOylated STAT4 was detected in BMDMs transduced with FLAG-tagged Stat4-WT but not Stat4-K350R after endogenous Stat4 was knocked down. (F) Western blotting was used to analyze nuclear STAT4 expression in macrophages transduced with FLAG-tagged Stat4-WT and Stat4-K350R adenoviruses after endogenous Stat4 was knocked down by siRNA. (G) Macrophages transduced with FLAG-tagged Stat4-WT and Stat4-K350R after knockdown of endogenous Stat4 were costained with anti-FLAG (red) and DAPI (blue) and imaged with confocal microscopy. (H) Transduced macrophages were pretreated with CHX for the indicated times, and STAT4 (FLAG-tagged) protein levels were analyzed. (I) Transduced macrophages were pretreated with CHX plus MG-132, and immunoprecipitation was conducted to identify ubiquitination level of STAT4 in the 2 groups. (J) Transcription levels of Ifn-g, Tnf-a, Cd86, and Mhc-i in virus-transduced macrophages quantified by real-time qPCR (n = 4 per group). (K) ELISA was conducted to check the secreted IFN-? and TNF-a of virus-transduced macrophages (n = 4 per group). (L) Macrophages transduced with Stat4-WT or Stat4-K350R after endogenous Stat4 knockdown were cocultured with CD8+ T cells in the presence of anti-CD3. The proportions of Ki67+ and IFN-?+ CD8+ T cells were examined (n = 4 per group). C–I represent at least 2 independent experiments. Data in J–L represent mean ± SEM and were analyzed by Student’s t test. **P < 0.01; ***P < 0.001; ****P < 0.0001.