Fig 2: CM from TRIM59-/--M2 contains TNF-a and induces expression of ccl2, MMP-12, MMP-9, and Madcam1 in melanoma cells. (A) ELISA detection of cytokines in culture supernatants from WT-M0, TRIM59-/--M0, WT-M2, and TRIM59-/--M2 macrophages. Data are represented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, compared with the TRIM59-/--M2 group. (B) Scatter plots showing DEGs detected in B16-F10 cells treated with TRIM59-/--M2 CM. (C) GO enrichment analysis of DEGs identified in B16-F10 cells exposed to TRIM59-/--M2 CM. (D) Top 15 DEGs. (E) qRT-PCR detection of the top 10 DEGs in cells treated with control media, WT-M2 CM, orTRIM59-/--M2 CM. Data are represented as mean ± SD. *p<0.05, compared with the WT-M2 CM group. (F) qRT-PCR detection of ccl2, MMP-12, MMP-9, and Madcam1 expression in B16-F0 and B16-F10 cells treated with or without TNF-a. Data are represented as mean ± SD. *p<0.05, **p<0.01, compared with untreated cells. (G) qRT-PCR detection of ccl2, MMP-12, MMP-9, and Madcam1 expression in B16-F0 and B16-F10 cells treated with control media, TRIM59-/--M2 CM, or TRIM59-/--M2 CM containing a neutralizing TNF-a antibody. Data are represented as mean ± SD. *p<0.05, **p<0.01, compared with untreated cells.

Fig 3: Knockdown of MMP-9 or Madcam1 inhibits migration, invasion, and EMT in melanoma cells. (A) Expression of MMP-9 and Madcam1 in B16-F0 and B16-F10 cells transfected with MMP-9-shRNA, Madcam1-shRNA or scrambled (control)-shRNA, as assessed by qRT-PCR. Data are represented as mean ± SD. **p<0.01, compared with control cells. (B) Expression of MMP9 and Madcam1 in B16-F0 and B16-F10 cells transfected with MMP-9-shRNA, Madcam1-shRNA, or scrambled (control)-shRNA, as assessed by western blotting. A representative image from three independent experiments is shown. (C) Representative images of cell migration assays conducted on B16-F10 cells transfected with control shRNA, MMP-9-shRNA, or Madcam1-shRNA. Data are represented as mean ± SD. *p<0.05, compared with control cells. (D) Representative images of transwell invasion assays conducted on B16-F10 cells transfected with control shRNA, MMP-9-shRNA, or Madcam1-shRNA. Data are represented as mean ± SD. *p<0.05, compared with control cells. (E) Western blot detection of EMT-associated proteins, including E-cadherin, N-cadherin, and vimentin, in B16-F0 and B16-F10 cells after shRNA-mediated downregulation of MMP-9 or Madcam1.

Fig 4: TRIM59-/--M2 macrophage CM activates the PI3K-Akt and ERK pathways in melanoma cells. (A) Western blot detection of signaling proteins related to the PI3K and ERK pathways in B16-F0 and B16-F10 cells treated with control media, WT-M2 CM, or TRIM59-/--M2 CM. Data are represented as mean ± SD. *p<0.05, **p<0.01, compared with the TRIM59-/--M2 CM group. (B) Western blot detection of signaling proteins related to the PI3K and ERK pathways in B16-F0 and B16-F10 cells treated with control media, TNF-a, or a TNF-a antibody. Data are represented as mean ± SD. *p<0.05, **p<0.01, compared with the TNF-a group. (C) Transwell assays results showing the invasive ability of B16-F0 and B16-F10 cells in response to CM from M2 macrophage cultures in the presence of the ERK inhibitor U0126. (D) Wound healing assay results showing the migratory ability of B16-F0 and B16-F10 cells in response to CM from M2 macrophage cultures in the presence of the ERK inhibitor U0126. (E) qRT-PCR evaluation of MMP-9 and Madcam1 expression in B16-F0 and B16-F10 cells exposed to CM from M2 macrophage cultures in the presence of the ERK inhibitor U0126. Data are represented as mean ± SD. *p<0.05, **p<0.01, WT-M2 CM vs. WT-M2 CM plus U0126; ?p<0.05, ??p<0.01, TRIM59-/--M2 CM vs. TRIM59-/--M2 CM plus U0126.

Fig 5: Knockdown of MMP-9 or Madcam1 inhibits melanoma growth in vivo. (A) Representative image of B16-F10 tumors from three independent experiments. WT or TRIM59-CKO mice were inoculated subcutaneously with B16-F10 cells transfected with control shRNA, MMP-9-shRNA, or Madcam1-shRNA. Tumor volumes were measured twice a week, and tumor weights estimated on sacrifice at day 28. n = 5 mice per group. Data are represented as mean ± SD. *p<0.05, **p<0.01, compared to tumors generated by shRNA control B16 cells in TRIM59-CKO mice. (B) Representative H&E staining images and IHC detection of Ki67, MMP-9, and Madcam1 in B16-F10 allografts (original magnification × 200). (C) Detection of MMP-9 and Madcam1 mRNA expression in B16-F10 allografts by qRT-PCR. Data are represented as mean ± SD. *p<0.05, **p<0.01, compared to tumors generated by shRNA control B16 cells in TRIM59-CKO mice.