Fig 1: PJA2 decreases KSR1 by ubiquitination, thus suppressing macrophage M2 polarization. A The expression of KSR1 in GC data collected by TCGA and GTEX (T: Tumor. N: normal. *p < 0.05). B KSR1 protein expression level in GC tissues and adjacent tissues of GC patients was detected by Western blot (n = 30). C KSR1 protein expression levels in TAMs in GC tissues and in Macs of adjacent tissues was detected by Western blot. D After overexpression of PJA2 in TAMs, Western blot was used to detect the protein expression of KSR1. E After overexpression of PJA2 in TAMs, the binding of KSR1 to ubiquitin was detected by co-IP assay. F The stability of KSR1 protein was detected by CHX treatment after overexpression of PJA2 in TAMs. G The mRNA and protein expression levels of M1 and M2 macrophage markers in TAMs cells were detected by RT-qPCR and ELISA. *p < 0.05
Fig 2: The molecular mechanism plot for the role of ELK4 in GC by regulating KDM5A/PJA2/KSR1 axis. ELK4 may inhibit PJA2 expression through transcriptional activation of KDM5A, which reduces the ubiquitination of KSR1, promoting M2 polarization of macrophages and thus facilitating the development of GC
Fig 3: KDM5A inhibits PJA2 expression by removing H3K4me3 and promoted macrophage M2 polarization. A The mRNA expression of PJA2 was detected by RT-qPCR (n = 30). B The mRNA expression of PJA2 in GC-TAMs and Macs of adjacent tissues was detected by RT-qPCR. C KDM5A protein expression was detected by Western blot after KDM5A was silenced in TAMs. D After KDM5A was silenced in TAMs, Western blot was used to detect the protein expression of PJA2. E After KDM5A was silenced in TAMs, the enrichment of H3K4me3 on PJA2 promoter was detected by ChIP assay. F The mRNA and protein expression of M1 and M2 macrophage markers in TAMs was detected by RT-qPCR and ELISA, respectively. *p < 0.05
Fig 4: ELK4 promotes M2 polarization of macrophages by regulating KSR1 to promote the proliferation, migration and invasion of GC cells. A Western blot was used to detect the expression of ELK4, KDM5A, PJA2 and KSR1 in TAMs. B mRNA and protein expression of M1 and M2 macrophage markers in TAMs cells was detected by RT-qPCR and ELISA. C The proliferation of HGC-27 cells was detected by CCK-8 assay after TAMs were co-cultured with HGC-27. D After TAMs were co-cultured with HGC-27 cells, the migration and invasion of HGC-27 cells were detected by Transwell assay (× 200). *p < 0.05
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