Peritoneal macrophage responses following GIFT15 treatment. (A) In vitro macrophage migration assay. Peritoneal macrophages were plated for 18 hours in transwell plates with lower chambers filled in triplicate with cytokines at increasing molarities. The cells on the bottom filters of 10 high-power fields (× 400) were counted for each well, and the results are depicted as mean cell number per high-power field ± SED. (B) STAT3/STAT5 phosphorylation in peritoneal macrophages. Macrophages (10⁶) were stimulated for 15 minutes with 30 pmol rIL-15, rGMCSF, or both cytokines inoculated in B16-GFP CM or with B16-GIFT15 CM, and cell lysate was probed for phosphorylated STAT3/STAT5. Total STAT3 or STAT5 protein was used as loading control. (C) TIMP-2 secretion from GIFT15-treated macrophages. Peritoneal macrophages treated with 30 pmol cytokines in serum-free media were incubated for 72 hours and media tested by angiogenic protein arrays (U87 supernatant was used as positive control). TIMP-2 detection was further confirmed by WB (+ control is either rMMP2 or rMMP9). (D) GIFT15 treatment induces the secretion and activation of MMP2. Macrophages (10⁶) were deprived of serum and cultured with 30 pmol cytokines. Gelatin zymography was performed, and the hydrolytic activity of MMP-2 was assessed. (E) A confirmation by WB was performed to verify that MMP-2 but not MMP-9 was indeed induced by GIFT15. (F) Macrophage treatments with GIFT15 induce active TGF-β. Serum-free supernatant collected from macrophages treated with 30 pmol cytokines was assessed for the presence of active TGF-β, and only the GIFT15 group led to its detection. (G) GIFT15-treated macrophages led to VEGF secretion. Stimulated peritoneal macrophages treated as previously mentioned were assessed by ELISA for the secretion of VEGF. Even though GMCSF led to modest secretion of VEGF, the GIFT15-treated group had a higher concentration than the remaining groups. Results are shown as mean ± SED (n = 3).