- PA5-47047 - Invitrogen Antibodies ADAM8 antibody

Submitted references The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness.

Schäfer A, Evers L, Meier L, Schlomann U, Bopp MHA, Dreizner GL, Lassmann O, Ben Bacha A, Benescu AC, Pojskic M, Preußer C, von Strandmann EP, Carl B, Nimsky C, Bartsch JW
Frontiers in oncology 2022;12:826273

ADAM8-Dependent Extracellular Signaling in the Tumor Microenvironment Involves Regulated Release of Lipocalin 2 and MMP-9.

Cook L, Sengelmann M, Winkler B, Nagl C, Koch S, Schlomann U, Slater EP, Miller MA, von Strandmann EP, Dörsam B, Preußer C, Bartsch JW
International journal of molecular sciences 2022 Feb 10;23(4)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry analysis of ADAM8 in immersion fixed human peripheral blood mononuclear cells (PBMCs). Samples were incubated in ADAM8 polyclonal antibody (Product # PA5-47047) using a dilution of 15 µg/mL for 3 hours at room temperature followed by NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 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 um. ( 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 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 mum. ( 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 DeltaCD 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 ADAM8 and LCN2 levels correlate in Panc89- and MB-231-derived extracellular vesicles (EV). Representative Western blots ( A ) of EVs derived from either Panc89 hA8 WT or KO cells, and cell lysate (CL) of Panc89 hA8 WT cells, and ( B ) of EVs derived from either MB-231 hA8 WT or MB-231 KO cells show the detection of ADAM8, MMP-9, LCN2, and Flotillin-1 in the upper part. Diagrams below illustrate the quantification and downregulation of LCN2 secretion (relative to Flotillin-1 secretion) in EVs isolated from Panc89 (hA8 WT or hA8 KO 1) or MB-231 (hA8 WT or hA8 KO) cells ( n = 3). Data are presented as mean values +- S.D. ** p < 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 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 mum. After co-culture, morphological changes are visible in both Panc89 hA8 WT and KO cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Co-culture of THP1-derived and polarized macrophages with Panc89 hA8 WT and KO cells. ( A ) Western blot illustrates the detection of ADAM8, MMP-9, and LCN2 in Panc89 hA8 WT and KO control cells (O) and after co-culture with M0, M1, and M2 macrophages (two time points: 0 h and 1 h). ADAM8 is upregulated in Panc89 hA8 WT cells after co-culture with M2-polarized macrophages. Panc89 cells show the highest MMP-9 expression after co-culture with M0, but M1 macrophages also upregulate MMP-9. LCN2 is dependent on ADAM8 when upregulated in Panc89 cells after co-culture with M0 and M2 macrophages but independent of ADAM8 in Panc89 cells co-cultured with M1 macrophages. ( B ) ADAM8, ( C ) MMP-9, and ( D ) LCN2 ELISA of Panc89 hA8 WT and KO cell-derived supernatants of control cells and after co-culture with M0, M1, and M2 (two time points: 0 h and 1 h). In accordance with the immunoblot results of ( A ), ADAM8 is upregulated in supernatants derived from Panc89 hA8 WT cells after co-culture with M2 macrophages ( B ). At the same time, macrophages increase MMP-9 secretion from an undetectable level to almost 80,000 pg/mL in Panc89 hA8 WT and 60,000 pg/mL in Panc89 hA8 KO cells. M1 and M2 macrophages increase MMP-9 secretion of Panc89 independent of ADAM8, but not as high as in Panc89 cells co-cultured with M0. In contrast, LCN2 is upregulated in Panc89 hA8 WT cells by M0 and M1, but not by M2 macrophages. In the absence of ADAM8, Panc89 hA8 KO cells show low LCN2 secretion in control cel

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Screening of two representative CRISPR/Cas9 ADAM8 (hA8) KO cell clones reveals an ADAM8-dependent regulation of miR-181a-5p. Confirmation of the CRISPR/Cas9 ADAM8 stable KO in U87 cells by qPCR (A) , Western Blot (B) , and ELISA (C) . (D) A RT-qPCR-based miRNA PCR Array (Human Finder, Qiagen) of U87 CRISPR/Cas9 ADAM8 KO cells enabling the analysis of a total of 84 miRNAs. The legend for the fold-changes in the heat map is given above representing the fold-change values (2 -DeltaDeltaCT ) relative to U87_CTRL cells in both ADAM8 KO clones. Note the variance in fold changes between the two KO clones (E) Confirmation of miR-181a-5p upregulation in U87_KO1 and U87_KO2 cells (p-value: 0.01 and 0.03). The expression of miR-181a-5p (F) and ADAM8 (G) in GBM cell lines (G28, G112, U251), primary patient-derived cell lines (GBM29, 98, and 42), and GBM stem-like cell lines (GSCs) (GSC 2016/240, GSC 2017/74, GSC 2017/151) are shown as relative values to the expression in U87 cells. MiR-181a-5p is mostly expressed in GSCs. The patient-derived cell line GBM42 shows the highest ADAM8 expression. Pearson correlation analysis of miR-181a-5p and ADAM8 in cell lines (H) , primary cell lines (I) , and primary GSCs (J) reveal a negative correlation only observed in GSCs (p-value: 0.01, Pearson r: -0.88). Results are given as mean +/- SD of two to three independent experiments. Unpaired two-tailed students t-test or two way ANOVA for multiple comparison (F) were applied to determine significance:

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 ADAM8 regulates the expression of miR-181a-5p via JAK2/STAT3 signaling. (A) U87_CTRL cells were analyzed for miR-181a-5p expression by RT-qPCR after treatment with the broad-spectrum MMP inhibitor BB-94 (left) and the ADAM8 inhibitor BK-1361 (right). (B) One representative western blot of three independent experiments shows the rescue of either ADAM8 lacking the cytoplasmatic domain (Delta CD) or the full-length ADAM8 (hA8). The quantifications of pEGFR, pSTAT3, and pERK1/2 are depicted on the right side and were normalized to beta-Tubulin and total-EGFR/beta-Tubulin, total STAT3/beta-Tubulin, or total ERK1/2/beta-Tubulin. Also, RT-qPCR results show no differences in miR-181a-5p expression after the transfection of ADAM8 Delta CD but a downregulation with the full-length ADAM8 rescue (p-value: 0.002). (C) The expression of ADAM8 mRNA (RT-qPCR, left) and secreted ADAM8 (ELISA, right, n=2) is not affected after miR-181a-5p mimic transfection. U87_CTRL cells (D) and patient-derived GBM42 cells (E) were treated with JAK2/STAT3 inhibitor WP1066 as indicated and analyzed via western blot and RT-qPCR. In (D) , qPCR results are shown as mean values +/- SD of four independent experiments and in (E) , results of miR-181a-5p are described as mean values of three technical replicates. Inhibition of JAK2/STAT3 increases miR-181a-5p expression (U87 p-value: 0.027; GBM42 p-value: 0.004). Results are shown as mean values +/- SD from three independent experiments if not otherwise sta

PA5-47047

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