Fig 1: Co-culture of THP1-derived macrophages with Panc89 hA8 WT and KO cells. (A) The schematic model depicts the interactions of THP1-derived macrophages (green, M0) and Panc89 cells with or without ADAM8 (red). Created with BioRender.com. (B) ADAM8 mRNA expression in both Panc89 hA8 WT and KO is not affected by M0, whereas LCN2 mRNA expression. Data are presented as mean values ± S.D. *** p < 0.001. (C) is upregulated after co-culture in an ADAM8-dependent manner. Data are presented as mean values ± S.D. *** p < 0.001. (D) The graph illustrates the upregulation of MMP-9 mRNA expression in both Panc89 hA8 WT and KO after co-culture (n = 2). Data are presented as mean values ± S.D. *** p < 0.001. (E) Representative immunoblot shows the detection of ADAM8, MMP-9, and LCN2 with or without co-culture. In addition to the qPCR results, MMP-9 and LCN2 are upregulated after co-culture at the protein level (n = 2). (F) Representative zymography of Panc89 hA8 WT and KO cells with or without co-culture demonstrates less active MMP-9 in Panc89 hA8 KO cells than in Panc89 hA8 WT cells after co-culture. (G) Quantification of active MMP-9 refers to total MMP-9 in zymography of Panc89 hA8 WT and KO cells after co-culture (n = 2). Data are presented as mean values ± S.D. * p < 0.05. Representative images of Panc89 cells before and after co-culture are shown in (H); scale bar, 100 μm. After co-culture, morphological changes are visible in both Panc89 hA8 WT and KO cells.
Fig 2: Downstream signaling of EGFR is affected by ADAM8 KO in Panc89 cells. (A) Representative immunoblot of EGFR and MAPK phosphorylation, and LCN2 expression after treatment with recombinant LCN2 (rLCN2) and recombinant transforming growth factor-alpha (rTGF-α) for 1 h. (B) Quantification of LCN2 shows the downregulation of LCN2 expression in Panc89 hA8 KO cells. The addition of rLCN2 does not increase LCN2 expression in Panc89 hA8 KO cells, whereas rTGF-α alone or combined with rLCN2 adjusts LCN2 expression to Panc89 hA8 WT levels. (C) Quantification of p-EGFR illustrates no significant changes in EGFR phosphorylation of Panc89 hA8 KO compared to Panc89 hA8 WT cells. (D) The diagram displays the quantification of p-MAPK. The phosphorylation of MAPK is significantly downregulated in Panc89 hA8 KO cells. rTGF-α stimulation decreases MAPK phosphorylation in both Panc89 hA WT and KO cells. Data are presented as mean values ± S.D. * p < 0.05, ** p < 0.01 (n = 2).
Fig 3: ADAM8 in Panc89 hA8 WT and KO cells. (A) mRNA expression, (B) Western blot, and (C) soluble ADAM8 levels (n = 2) in Panc89 hA8 WT and KO 1 and 2. (D) Representative immunofluorescence (green) of ADAM8 in Panc89 hA8 WT and KO cells; scale bar, 20 µm. (A–D) show the successful downregulation of ADAM8 in KO 1 and 2 cells. (E,F) display scratch assay of Panc89 hA8 WT and KO 1 cells. Images were acquired at 0 h and 10 h (n = 2). (G) Invasion assay of Panc89 hA8 WT and KO cells in Matrigel using transwell inserts (24 h) demonstrates a decreased invasive behavior in KO cells (n = 3). (H) Relative growth rates of Panc89 cells show no significant differences between hA8 WT, KO 1, and KO 2 cells (n = 2). MMP and ADAM activity assays of Panc89 hA8 WT- and KO cell-derived supernatants (SN) by using PepDAB# (I) 5, (J) 8, (K) 10, (L) 13, and (M) 14 are illustrated (n = 2). (N) Heat map of mean Ct values demonstrates the absolute gene expression of MMP-2, MMP-9, and ADAM17, and (O) diagram shows the relative mRNA expression of MMP-2 and ADAM17 in Panc89 hA8 WT and KO 1 cells (n = 2). (P,Q) show results of protease activities and cleavage rates of MMP-2, MMP-9, ADAM8, and ADAM17 calculated for hA8 WT and KO 1 cell-derived supernatants by PrAMA inference. Data are presented as mean values ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 4: ADAM8 regulates LCN2 levels in tumor cell lines MB-231 and Panc89. The Human Protease/Protease Inhibitory Array demonstrates downregulation of ADAM8 (A), MMP-9 (B), and LCN2 (C) in supernatants derived from MB-231 hA8 KO cells. (D) mRNA expression and (E) representative Western blot of LCN2 in MB-231 hA8 WT and hA8 KO cells confirm results from (C) (n = 2). (F) mRNA expression and (G) representative Western blot of LCN2 in Panc89 hA8 WT, KO 1, and KO 2 cells demonstrate decreased LCN2 expression in hA8 KO cells (n = 2). Data are presented as mean values ± S.D., ** p < 0.01, *** p < 0.001. (H) Recombinant LCN2 (1 ng, 10 ng, 100 ng) does not affect protease activity of recombinant ADAM8 using a CD23 substrate (PepDAB# 13).
Fig 5: ADAM8 is secreted by Panc89 hA8 WT-derived extracellular vesicles (EV). (A) The histogram shows the particle size distribution of EVs isolated from Panc89 cells (analyzed by NanoFCM). (B) Electron microscopy of Panc89 hA8 WT-derived EVs demonstrates the successful isolation of EVs; scale bar, 100 nm. Representative Western blot of EVs derived either from Panc89 hA8 WT or KO cells, and cell lysate (CL) of Panc89 hA8 WT cells is shown in (C). ADAM8 can be detected as active and remnant ADAM8 in EVs. The negative control Calnexin was not detectable in isolated EVs. The measured activity of Panc89 hA8 WT- and KO-derived EVs on PepDAB #13 is displayed in (D) and is upregulated in Panc89 hA8 WT-derived EVs (n = 2). (E) Representative images of immunofluorescence staining of Panc89 hA8 rescue cells with Hoechst dye (upper left), TSG101 (green; upper right), and ADAM8 (red; lower left). Merged images are displayed in the lower right and show that TSG101 shows little or no co-localization with ADAM8. Scale bar, 50 μm. (F) shows the detection of ADAM8, Flotillin-1, and CD81 via Western blot of Panc89 hA8 WT CL and EV preparations isolated from Panc89 hA8 WT, Panc89 hA8 rescue, Panc89 hA8 ΔCD rescue, and Panc89 hA8 KO cells. ADAM8 is detectable in all EV preparations except in EVs isolated from hA8 KO cells. Data are presented as mean values ± S.D.
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