Fig. 6
Decipher the signaling interaction between macrophage and ERKactiveDUSP2low epithelial cells. (A) Circle plot showing the inferred signaling networks from C15-Epi subset toward subsets of other epithelial, macrophage and T cells in early and advanced stages. The round loops along with cell type represent the interactions within the same cell type. (B) Circle plots showing the outgoing signals from each macrophage subset (C4, C9, and C12) in the early PDAC. (C) Hallmark pathway enrichment analysis of DEGs in C9-Mac in early and advanced PDAC tumor. (D) Cytokine array (RayBiotech) was used to identify potential factors secreted from macrophage (MCM) compared to RPMI which contains 10% FBS. (E) The expression of TIMPs in macrophages. Macrophages were first co-cultured with PANC-1 for 2 days and then cultured alone by serum-free RPMI medium for an additional day. Conditioned medium was collected and concentrated to measure the expression of TIMP-1 and TIMP-2 by Western blotting. (F) PANC-1 cells were initially treated with MCM for 1 h, after which they were cultured in serum-free medium with or without recombinant TIMP-1 (Peprotech) for 24 h. Western blotting was then performed to assess the expression of DUSP2, pERK, E-cadherin, and β-actin. (G) The expression of CD63, pERK, tERK, DUSP2, and internal control β-actin in control and CD63 knockdown (KD) PANC-1 cells after being treated with MCM for 1 h. (H) Control and CD63-KD PANC-1 cells were first treated with or without MCM for 24 h and the medium was replaced by serum-free RPMI for an additional 24 h. The expression of CD63, pERK, tERK, DUSP2, and internal control β-actin was determined by Western blotting. (I) The volcano plot illustrates the number of genes that are significantly upregulated or downregulated in MCM-treated cells compared to untreated cells under both Ctrl-KD and CD63-KD conditions, with a fold change greater than 2. (J) GO analysis was conducted on differentially expressed genes comparing MCM-treated Ctrl-KD and MCM-treated CD63-KD cells, focusing on their involvement in biological processes and molecular functions, as illustrated in cnetplots and dotplots. (K) The expression of CD63, pERK, and internal control β-actin in PANC-1 cells that have been co-cultured with monocyte (co-U) for 3 days, with macrophage (co-M) for 2 days, or treated with MCM for 2 days. (L) The expression of CD63 and β-actin in control and DUSP2-KD PANC-1 cells treated with DMSO or ERK inhibitor (SCH772984)