Journal: Molecular medicine reports

Article Title: TrkB/C-induced HOXC6 activation enhances the ADAM8-mediated metastasis of chemoresistant colon cancer cells.

doi: 10.3892/mmr.2021.12062

Figure Lengend Snippet: Figure 2. Expressions of HOXC6 and ADAM8 are significantly increased in chemoresistant colon cancer cells. OxR or FuR colon cancer and parent cells were seeded into 6‑well plates (1.5x105/well) and cultured for 24 h. (A and C) Quantitative real‑time PCR was performed to determine the relative expression of HOXC4, HOXC6, ADAM8, ADAM10, ADAM12 and ADAM17. (B and D) The total protein of each group was subjected to western blot analysis with the indicated antibodies. β‑actin served as an internal control. (E) The migration and invasion of cells were detected using tumor transendothelial migration and BME cell invasion assay kits, respectively. (F) The gelatinase activity of cells was measured using a gelatinase assay kit. Data are presented as the mean ± SD of three independent experiments. *P<0.05, #P<0.005 and ##P<0.001 vs. resistant cells. HOX, homeobox; ADAM, A disintegrin and metallopro teinase domain‑containing 8; OxR, oxaliplatin resistant; FuR, 5‑Fu‑resistant; p, phosphorylated.

Article Snippet: The following primary antibodies were used: TrkB (#4603), TrkC (#3376), phospho‐ERK1/2 (Thr202/Tyr204; #9101), ERK1/2 (#9102), MMP2 (#4022), MMP9 (#3852), E‐cadherin (#3195), N‐cadherin (#13116), Snail (#3879), and β‐actin (#4967) (Cell Signaling Technology); phospho‐TrkB (Tyr817; #NBP2‐67578), ADAM8 (#NB600‐1393), and HOXC4 (#NBP2‐56195) (Novus Biologicals); phospho‐TrkC (Tyr518; #PA5‐40271) (Thermo Fisher Scientific); and HOXC4 (#sc‐81965), HOXC6 (#sc‐376330), phospho‐MEK1/2 (Ser218/Ser222; #sc‐7995), and MEK1/2 (#sc‐436) (Santa Cruz Biotechnology).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control, Migration, Invasion Assay, Activity Assay

Journal: Molecular medicine reports

Article Title: TrkB/C-induced HOXC6 activation enhances the ADAM8-mediated metastasis of chemoresistant colon cancer cells.

doi: 10.3892/mmr.2021.12062

Figure Lengend Snippet: Figure 4. MEK/ERK activation induces HOXC6 and ADAM8 during chemoresistant colon cancer cell metastasis. OxR or FuR colon cancer cells were seeded into 6‑well plates (1.5x105/well) and treated with 20 µM PD98059 for 24 h. (A and B) Total cell lysates were immunoblotted with the indicated antibodies. β‑actin served as an internal control. (C) The gelatinase activity of cells was measured using the gelatinase assay kit. (D) The migration and invasion of cells was detected using tumor transendothelial migration and BME cell invasion assay kits, respectively. Data are presented as the mean ± SD of three independent experiments. *P<0.05, **P<0.01, #P<0.005 and ##P<0.001 as indicated. HOX, homeobox; ADAM, A disintegrin and metalloproteinase domain‑containing 8; OxR, oxaliplatin resistant; FuR, 5‑Fu‑resistant; Trk, tropomyosin receptor kinase; p, phosphorylated.

Article Snippet: The following primary antibodies were used: TrkB (#4603), TrkC (#3376), phospho‐ERK1/2 (Thr202/Tyr204; #9101), ERK1/2 (#9102), MMP2 (#4022), MMP9 (#3852), E‐cadherin (#3195), N‐cadherin (#13116), Snail (#3879), and β‐actin (#4967) (Cell Signaling Technology); phospho‐TrkB (Tyr817; #NBP2‐67578), ADAM8 (#NB600‐1393), and HOXC4 (#NBP2‐56195) (Novus Biologicals); phospho‐TrkC (Tyr518; #PA5‐40271) (Thermo Fisher Scientific); and HOXC4 (#sc‐81965), HOXC6 (#sc‐376330), phospho‐MEK1/2 (Ser218/Ser222; #sc‐7995), and MEK1/2 (#sc‐436) (Santa Cruz Biotechnology).

Techniques: Activation Assay, Control, Activity Assay, Migration, Invasion Assay

Journal: Molecular medicine reports

Article Title: TrkB/C-induced HOXC6 activation enhances the ADAM8-mediated metastasis of chemoresistant colon cancer cells.

doi: 10.3892/mmr.2021.12062

Figure Lengend Snippet: Figure 5. HOXC6 knockdown in OxR or FuR colon cancer cells decreases cell invasion and downregulates ADAM8 activation and gelatinase activity. OxR or FuR colon cancer cells were seeded into 6‑well plates (1.5x105/well) and grown overnight. Cells were transfected with small interfering RNA against HOXC6 or control for 48 h. (A) Total cell lysates were immunoblotted with the indicated antibodies. β‑actin served as an internal control. (B) The gelatinase activity of cells was measured by the gelatinase assay kit. (C) The migration and invasion of cells was detected using tumor transendothelial migration and the BME cell invasion assay kits, respectively. Data are presented as the mean ± SD of the three independent experiments. *P<0.05, **P<0.01, #P<0.005 and ##P<0.001 as indicated. HOX, homeobox; OxR, oxaliplatin resistant; FuR, 5‑Fu‑resistant; ADAM, A disintegrin and metalloproteinase domain‑containing 8; Trk, tropomyosin receptor kinase; si, small interfering RNA; ctrl, control; p, phosphorylated.

Article Snippet: The following primary antibodies were used: TrkB (#4603), TrkC (#3376), phospho‐ERK1/2 (Thr202/Tyr204; #9101), ERK1/2 (#9102), MMP2 (#4022), MMP9 (#3852), E‐cadherin (#3195), N‐cadherin (#13116), Snail (#3879), and β‐actin (#4967) (Cell Signaling Technology); phospho‐TrkB (Tyr817; #NBP2‐67578), ADAM8 (#NB600‐1393), and HOXC4 (#NBP2‐56195) (Novus Biologicals); phospho‐TrkC (Tyr518; #PA5‐40271) (Thermo Fisher Scientific); and HOXC4 (#sc‐81965), HOXC6 (#sc‐376330), phospho‐MEK1/2 (Ser218/Ser222; #sc‐7995), and MEK1/2 (#sc‐436) (Santa Cruz Biotechnology).

Techniques: Knockdown, Activation Assay, Activity Assay, Transfection, Small Interfering RNA, Control, Migration, Invasion Assay

Journal: Molecular medicine reports

Article Title: TrkB/C-induced HOXC6 activation enhances the ADAM8-mediated metastasis of chemoresistant colon cancer cells.

doi: 10.3892/mmr.2021.12062

Figure Lengend Snippet: Figure 6. Activation of ADAM8 but not ADAM10 or ADAM17 regulates the invasion and gelatinase activity of metastatic drug‑resistant colon cancer cells. OxR or FuR colon cancer cells were seeded into 6‑well plates (1.5x105/well) and grown overnight. Cells were transfected with small interfering RNA against ADAM8 or control for 48 h, or treated with GI (10 µM) and MA (50 nM) for 24 h. (A and B) Total cell lysates were immunoblotted with the indicated antibodies. β‑actin served as an internal control. (C) The gelatinase activity of cells was measured using the gelatinase assay kit. (D) The migration and invasion of cells were detected using tumor transendothelial migration and BME cell invasion assay kits, respectively. Data are presented as the mean ± SD of three independent exper iments. *P<0.05 and ##P<0.001 as indicated. (E) Schematic diagram of the intracellular signaling pathway in drug‑resistant human colon cancer cells. ADAM8 expression contributed to the increased resistance and migratory activity of chemoresistant colon cancer cells through the activation of the TrkB/C‑HOXC6 axis. ADAM, A disintegrin and metalloproteinase domain‑containing 8; OxR, oxaliplatin resistant; FuR, 5‑Fu‑resistant; GI, ADAM10 inhibitor GI254023X; MA, ADAM17 inhibitor marimastat; Trk, tropomyosin receptor kinase; HOX, homeobox; si, small interfering RNA; Ctrl, control; p, phosphorylated.

Article Snippet: The following primary antibodies were used: TrkB (#4603), TrkC (#3376), phospho‐ERK1/2 (Thr202/Tyr204; #9101), ERK1/2 (#9102), MMP2 (#4022), MMP9 (#3852), E‐cadherin (#3195), N‐cadherin (#13116), Snail (#3879), and β‐actin (#4967) (Cell Signaling Technology); phospho‐TrkB (Tyr817; #NBP2‐67578), ADAM8 (#NB600‐1393), and HOXC4 (#NBP2‐56195) (Novus Biologicals); phospho‐TrkC (Tyr518; #PA5‐40271) (Thermo Fisher Scientific); and HOXC4 (#sc‐81965), HOXC6 (#sc‐376330), phospho‐MEK1/2 (Ser218/Ser222; #sc‐7995), and MEK1/2 (#sc‐436) (Santa Cruz Biotechnology).

Techniques: Activation Assay, Activity Assay, Transfection, Small Interfering RNA, Control, Migration, Invasion Assay, Expressing

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A ADAM8 mRNA expression in samples from breast tumor and normal breast tissue was analyzed using the Oncomine microarray database. Pooling of 14 analyses from six different microarray studies shows ADAM8 is one of the more highly expressed genes in breast cancer versus normal tissue. P = 0.025, Student's t -test. B, C ADAM8 protein levels were measured by ELISA in samples from adjacent normal breast tissue (NBT), fibroadenoma (FA) and primary breast carcinoma (PBC) (B), and in serum of patients with either benign or malignant breast disease (C). * P < 0.0001, Kruskal–Wallis test (B); * P = 0.034, Mann–Whitney U -test (C). D ADAM8 mRNA expression was analyzed across the different molecular breast cancer subtypes in the van de Vijver microarray dataset, which includes 295 primary breast tumors from normal-like (Normal), luminal A (Lum A), luminal B (Lum B), HER2, and basal-like (Basal) subtypes (van de Vijver et al , ). * P < 0.0001 for Basal versus other groups, Kruskal–Wallis test. E Representative pictures of ADAM8 staining analyzed by immunohistochemistry in adjacent normal epithelial tissue and primary TNBC samples from 50 patients or areas of microinvasion. Percentages of ADAM8-positive samples are given. F, G Kaplan–Meier curves show the percentage of disease-free survival (F) and overall survival (G) for 295 patients with primary breast cancer stratified based on ADAM8 mRNA levels using the 75th percentile. P < 0.0001, Log-rank test (RR, relative risk, CI, confidence interval).

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Expressing, Microarray, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Staining, Immunohistochemistry

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A Schematic representation of ADAM8 protein with its domains, processed forms and molecular weights indicated. CYS-Rich: cysteine-rich, EGF: EGF-like, TM: transmembrane domains. B Whole-cell extracts (WCEs) from human non-tumoral MCF-10A cells and the indicated TNBC cell lines were examined by WB for ADAM8 expression (Millipore antibody), and for β-Actin as a loading control. A representative blot is shown ( n = 3). All lanes were from the same gel, but cut to re-align as indicated by the vertical line. ADAM8 forms and MW markers are indicated. ns: non-specific band. MDA-231: MDA-MB-231. MDA-468: MDA-MB-468. C Cells were grown in adherent (2D) or suspension cultures on ultra low-attachment plates (3D) for 48 h. In suspension, MDA-MB-231 and Hs578T cells form spheres. Bar: 100 μm. D, E MDA-MB-231 (D) and Hs578T (E) cells were transfected with either Control siRNA (siCtrl) or two specific ADAM8 siRNAs (siA8) for 24 h. Cells were then plated in 2D-or 3D-culture as described in (C). WCEs were subjected to WB for ADAM8 using only LSBio antibody (D), or using both LSBio and Millipore antibodies (E). As a loading control, Tubulin (D) or β-Actin (E) was used. Representative blots are shown ( n = 3).

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Expressing, Control, Suspension, Transfection

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A–D Cells were transfected with siRNAs as in Fig D and E for 24 h, and tested for colony formation in soft agar (A), cell migration (B), invasion (C), and 3D-Matrigel outgrowth (D). For soft agar assays, cells were grown for 8-12 days and colonies >20 μm diameter in 3 wells/condition were counted with ImageJ software. A representative of two experiments with similar results is shown. * P = 0.002, ** P = 1.9E-6, # P = 1.3E-5, ## P = 1.0E-7, Student's t- test (A). Migration (B) and invasion (C) assays were performed for 24 h using Transwells without or with precoating of Matrigel, respectively. Control condition (siCtrl) is set to 100% (mean ± s.d. from three independent experiments). * P = 0.004, ** P = 5.3E-4, # P = 1.8E-4, ## P = 5.5E-5, Student's t- test (B). * P = 0.001, ** P = 9.3E-4, # P = 8.7E-6, ## P = 4.6E-5, Student's t- test (C). For Matrigel outgrowth assays, colonies formed after 5-7 days were photographed at 20× magnification. Experiments were done twice with similar results. Bars: 100 μm (D). E WCEs from MDA-MB-231 cells and in vivo derived LM1 and LM2 lines, either untransfected (left panel) or transfected with siCtrl or siA8 for 48 h (right panels), were subjected to WB for ADAM8 (LSBio antibody). A representative blot is shown ( n = 3). F Twenty-four h after siRNA transfection, LM1 and LM2 cells were subjected to a 3D-Matrigel outgrowth assay, as in (D), except that colonies were photographed after 4 days. Experiments were done twice with similar results. Bars: 100 μm.

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Transfection, Migration, Software, Control, In Vivo, Derivative Assay

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A–D Stable ADAM8 KD (shA8) clones were characterized in vitro . ADAM8 expression in WCEs from two shA8 clones (shA8-17 and shA8-20) were compared with two shCtrl clones (shCtrl-3 and shCtrl-5) using WB (LSBio antibody) (A). Clones were grown in 2D-cultures for 48 h and subjected to an ATP assay. NS: non significant, Student's t -test, n = 3 (B). Migration assays were performed as described in Fig B. * P = 6.8E-14, Student's t- test, n = 3 (C). Matrigel outgrowth assays were performed as in Fig D. Experiments were done twice with similar results. Bar: 100 μm (D). E–H MDA-MB-231 derived shCtrl-3 and shA8-20 cells were injected into the MFP of female mice ( n = 7/group). Tumor volume was measured twice a week (mean ± s.e.m.). * P = 1.4E-6, ** P = 9.8E-8, # P = 3.0E-5, ## P = 9.7E-7, † P = 1.7E-5, ‡ P = 5.1E-6, § P = 2.0E-7, Student's t -test (E). At the end of the experiment, tumors were photographed and weighed (mean ± s.e.m.). * P = 5.6E-8, Student's t -test. Bar: 1 cm (F). Blood was collected by cardiac puncture and GFP-positive CTCs were detected by flow cytometry. Average CTC count ± s.d. is given per μl of blood. * P = 0.006, Student's t -test (G). Presence of brain metastases was examined by fluorescent microscopy ( n = 6/group). Representative photographs are shown. Bars: 1 mm (H). I ADAM8 expression in distant metastases of breast cancer patients ( n = 56) was analyzed by immunohistochemistry. Representative pictures and percentages of ADAM8-positive samples are presented.

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Clone Assay, In Vitro, Expressing, ATP Assay, Migration, Derivative Assay, Injection, Flow Cytometry, Microscopy, Immunohistochemistry

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A Sixteen h after plating, cells were cultured under normoxic (−) or hypoxic (+, 1% O2) conditions for 24 h (MDA-MB-231 and Hs578T) or 6 h (shCtrl-3 and shA8-20 clones). WCEs were subjected to WB for ADAM8 (LSBio antibody). Representative blots are shown ( n = 3). B ADAM8 expression in mouse mammary tumors derived from shCtrl-3 or shA8-20 cells was analyzed by immunohistochemistry. H&E staining was performed in parallel. Representative panels are shown ( n = 7/group). Bar: 100 μm. C Angiogenesis was evaluated by CD31 immunohistochemical staining of tumor sections from shCtrl-3 and shA8-20 groups ( n = 7/group). Vessel density for each mouse is given as the average number of vessels in 2 slides/tumor (3 peritumoral hot spots/slide). P: Peritumoral area, T: Tumor. Bar: 100 μm. * P = 0.01, Student's t -test. D Pearson's pairwise correlation plot shows a significant positive correlation between ADAM8 and CD31 (PECAM1) mRNA expression in tumors from patients with basal-like breast cancer (GenExMiner microarray database). r: correlation ratio. P < 0.0001, Student's t -test. E, F HUVECs were subjected to tube formation assays in the presence of conditioned medium from the indicated shA8 and shCtrl clones, or obtained in absence of tumor cells (−). Values for branch points and closed networks (polygons) are given as averages of nine fields ± s.d. Branch point: * P = 1.3E-6 versus shCtrl-3, * P = 3.2E-9 versus shCtrl-5, ** P = 6.9E-29; Polygons: * P = 5.2E-6 versus shCtrl-3, * P = 1.3E-6 versus shCtrl-5, ** P = 6.2E-24; Student's t -test; n = 4 (E). Representative images from 4 independent experiments are shown. Bar: 30 μm (F). G Conditioned media from two shCtrl and two shA8 clones were subjected to a Human Angiogenesis antibody array. Expression levels of the detected proteins were quantified using ImageJ and the angiogenesis mediators significantly downregulated by more than 2-fold in shA8 clones are presented as mean of the two clones ± s.d. Fold change (F.C.) and P -values are given, Student's t -test. H Conditioned serum-free medium from shCtrl and shA8 clones was analyzed by WB for VEGF-A. A representative blot is shown ( n = 3). I VEGF-A in the conditioned serum-free medium from shCtrl-3 or shA8-20 clones transfected with the indicated ADAM8 forms or empty vector (EV) DNA was assessed by WB (lower panel). The quantification of average levels from 3 experiments is presented as percent relative to the shCtrl set to 100%.

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Cell Culture, Clone Assay, Expressing, Derivative Assay, Immunohistochemistry, Staining, Immunohistochemical staining, Microarray, Ab Array, Transfection, Plasmid Preparation

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A Blood was drawn using the submandibular collection method from 4 mice/group on the indicated days after tumor cell implantation into the MFP and subjected to flow cytometry to measure GFP-positive CTCs. Average count for the 4 mice ± s.d. per μl of blood is given for each time point. * P = 0.03, # P = 0.05, † P = 0.02, Student's t -test. B Cells were incubated, in duplicate, on a HUVEC monolayer or in empty wells. Attached cells were counted in three fields per well. Mean ± s.d. from three independent experiments. NS, non significant, * P = 1.6E-6, Student's t -test. C Cells were subjected to an overnight transendothelial migration assay on HUVECs. Mean ± s.d. from three independent experiments. * P = 1.7E-5, Student's t -test. D, E Cells were plated on fibronectin-coated coverslips, stained with antibodies against active β1-integrin, vinculin or phosphotyrosine (focal adhesions markers) and phalloidin-488 (F-actin). Confocal microscopy images show adhesion protein channel (red) or merge with F-actin (green). Bar: 10 μm (D). Signal intensity for active β1-integrin and vinculin was determined using ImageJ software (arbitrary units). Mean intensity ± s.d. from >20 cells over 2 experiments. * P = 1.4E-4, NS, non significant, Student's t -test (E). F Adhesion of shCtrl-3 cells on HUVECs was assessed as in (B) with prior incubation of shCtrl-3 cells with an antagonist β1-integrin antibody (+) or a control isotype (−). Mean ± s.d. from three independent experiments. * P = 3.0E-5, Student's t -test. G, H Adhesion (G) and transendothelial migration (H) of shCtrl-3 cells on HUVECs was assessed as in B and C, respectively. Prior to the assays, shCtrl-3 cells were incubated either with monoclonal antibodies targeting the ectodomain of ADAM8 (Mab10311 or Mab1031) or an appropriate isotype control (IgG1 or IgG2B, respectively). The transmigration assay was performed for 9 h. Mean ± s.d. from three independent experiments. * P = 0.001, # P = 5.0E-4, Student's t -test (G). * P = 6.3E-7, # P = 4.0E-7, Student's t -test (H). I Active β1-integrin expression was assessed by immunohistochemistry in mouse mammary tumors (shCtrl-3 and shA8-20) and contralateral mammary glands (shCtrl-3) ( n = 7/group). P: Peritumoral area, T: Tumor. Bar: 100 μm.

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Flow Cytometry, Incubation, Migration, Staining, Confocal Microscopy, Software, Control, Bioprocessing, Transmigration Assay, Expressing, Immunohistochemistry

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: A–E MDA-MB-231 shCtrl-3 cells were injected into the MFP of female mice. Animals were treated with either 0.5 mg/kg anti-ADAM8 (anti-A8, Mab1031, n = 9) or isotype-matched control (IgG2B, n = 8) in i.p. injection twice weekly. Tumor volume was measured on the indicated days (mean ± s.e.m.). * P = 0.02 (day 11), # P = 0.01 (day 13), † P = 2.1E-4 (day 17), ‡ P = 1.5E-4 (day 20), Student's t -test (A). At the end of the experiment, tumors were weighed (mean ± s.e.m.). * P = 5.8E-4, Student's t -test (B) and the presence of brain metastases was examined by fluorescent microscopy (C). Angiogenesis was evaluated by CD31 immunohistochemical staining of tumor sections. Vessel density for each mouse is given as the average number of vessels in 2 slides/tumor (5 peritumoral hot spots/slide) and representative pictures are shown (D). P: Peritumoral area, T: Tumor. Bar: 100 μm. VEGF-A levels in the tumor extracts were determined by WB and normalized to Coomassie staining (E). * P = 0.04 (D), * P = 0.03 (E), Student's t -test. F–G Scheme of experimental design (F). Metastases to the brain and lungs were examined by fluorescent microscopy. Representative images and frequency of metastases (percentage of animals positive per group: IgG2B, n = 8; anti-A8, n = 9) are presented (G). Bar: 250 μm.

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Injection, Control, Microscopy, Immunohistochemical staining, Staining

Journal: EMBO Molecular Medicine

Article Title: ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

doi: 10.1002/emmm.201303373

Figure Lengend Snippet: When solid tumors reach a few millimeters in diameter, hypoxic stress is induced, which leads to ADAM8 induction resulting in strong pro-angiogenic signaling, in part via VEGF-A release into the extracellular compartment, and endothelial cell recruitment. ADAM8 also promotes β1-integrin activation on tumor cells needed for their adhesion onto and transmigration through the blood vessel wall, which supports dissemination of CTCs and development of metastases. Importantly, here we demonstrate that if the induction of ADAM8 is blocked or its activity inhibited with antibody, there is an insufficient angiogenic response, leading to tumor mass dormancy or slowing of growth, as well as to a striking reduction of CTCs and metastases.

Article Snippet: For inhibition experiments with antibodies, tumor cells were pre-treated with 10 μg/ml of β1-integrin antibody (BD Biosciences, 552828), 20 μg/ml of ectodomain ADAM8 antibody (Mab1031 and Mab10311; R&D Systems, Minneapolis, MN, USA), or control isotypes (rat or mouse, respectively) for 30 min at room temperature and then washed twice with PBS before being used.

Techniques: Activation Assay, Transmigration Assay, Activity Assay

Journal: Scientific Reports

Article Title: A novel peptide ADAM8 inhibitor attenuates bronchial hyperresponsiveness and Th2 cytokine mediated inflammation of murine asthmatic models

doi: 10.1038/srep30451

Figure Lengend Snippet: Lung tissue sections were stained by hematoxylin & eosin (H&E) shown in panel a–c, and immunohistochemistry (IHC) shown in panel d–f to visualize bronchiolar structure and ADAM8 expression, respectively. Panel g-i display zoom-in images corresponding to panel d–f, with arrows pointing to ADAM8-positive eosinophils. The bar in panel a-f equals 25 μm, whereas the bar in panel g-i equals 12.5 μm. The left, middle, and right column of images represents lung tissue sections from control group (panel a, d, g), OVA group (panel b, e, h), and OVA/BK-1361 group (panel c, f, i), respectively. Note the OVA-induced thickening of bronchiolar epithelium compared to control (panel b vs. a), which was reduced by BK-1361 treatment (panel c vs. b), and the OVA-induced fibrotic changes (panel h vs. g), which was also reduced by BK-1361 treatment (panel i vs. h). ADAM8-positive eosinophils (ADAM8 + cells) appeared mainly in OVA group (panel h vs. g), and became much less abundant after BK-1361 treatment (panel i vs. h). Panel j, k displays quantified bronchial wall thickness, and number of ADAM8 + cells, respectively. Data are representative of two independent experiments with n = 30–40 counts from 3–4 age-matched female mice per group. Result are expressed as mean ± SD. **P < 0.005 versus control. ## P < 0.005 versus OVA.

Article Snippet: Co. (Nanjing, China); TRIzol solution was purchased from Bioteke Co. (Beijing, China); RevertAid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA); Oligonucleotide primers were produced by Invitrogen Life Technologies Co. (Shanghai, China); SsoFast EvaGreen Supermix was purchased from Bio-Rad Laboratories (Hercules, CA, USA); ELISA kits for soluble CD23 (sCD23) and soluble ADAM8 (sADAM8) was purchased from either Life Science Inc. (Wuhan, China)or R&D Systems (Wiesbaden, Germany); NanoDrop was purchased from PeqLab (Erlangen, Germany); the ADAM8 antibody (Cat#: orb4376) was purchased from Biorbyt (Cambridge, U.K.), CD23 antibody (abb185807) from Abcam (Cambridge, U.K.), goat anti-mouse HRP and goat anti-rabbit HRP from Jackson ImmunoResearch (West Grove, PA, USA), mouse anti-GAPDH from Thermo Fisher Scientific (Waltham, MA, USA); VECTASTAIN Elite ABC Kit was purchased from Vector Laboratories (Peterborough, U.K.).

Techniques: Staining, Immunohistochemistry, Expressing, Control

Journal: Scientific Reports

Article Title: A novel peptide ADAM8 inhibitor attenuates bronchial hyperresponsiveness and Th2 cytokine mediated inflammation of murine asthmatic models

doi: 10.1038/srep30451

Figure Lengend Snippet: Total CD23 in lung tissue lysates and soluble CD23 (sCD23) in soluble protein fraction detected via ELISA are presented in panel a, and b respectively. The WB analysis results of sCD23 in lung tissue lysates and the representative image ofWB bands of CD23 are displayed in panel c, and d, respectively. Note the band for sCD23 around 38 kDa. For the quantification, values are derived from n = 3 (wild-type) and n = 4 (ADAM8-deficient) and are given as mean ± SEM. *p < 0.05 versus litter treated with saline.

Article Snippet: Co. (Nanjing, China); TRIzol solution was purchased from Bioteke Co. (Beijing, China); RevertAid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA); Oligonucleotide primers were produced by Invitrogen Life Technologies Co. (Shanghai, China); SsoFast EvaGreen Supermix was purchased from Bio-Rad Laboratories (Hercules, CA, USA); ELISA kits for soluble CD23 (sCD23) and soluble ADAM8 (sADAM8) was purchased from either Life Science Inc. (Wuhan, China)or R&D Systems (Wiesbaden, Germany); NanoDrop was purchased from PeqLab (Erlangen, Germany); the ADAM8 antibody (Cat#: orb4376) was purchased from Biorbyt (Cambridge, U.K.), CD23 antibody (abb185807) from Abcam (Cambridge, U.K.), goat anti-mouse HRP and goat anti-rabbit HRP from Jackson ImmunoResearch (West Grove, PA, USA), mouse anti-GAPDH from Thermo Fisher Scientific (Waltham, MA, USA); VECTASTAIN Elite ABC Kit was purchased from Vector Laboratories (Peterborough, U.K.).

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Saline

Journal: BMC Cancer

Article Title: Expression of A disintegrin and metalloprotease 8 is associated with cell growth and poor survival in colorectal cancer

doi: 10.1186/1471-2407-14-568

Figure Lengend Snippet: Primers used for qRT-PCR

Article Snippet: After deparaffinization in xylene and rehydration through a graded alcohol series, slides were transferred to sodium citrate buffer (Beijing DingguoChangsheng Biotech Co. Ltd, AR-0511) for 15 min in the microware and left at room temperature for 30 min. After blocking endogenous peroxidase, slides were incubated with 10 μg/ml goat polyclonal antibody specific to human ADAM8 (R&D Systems, Inc., Minneapolis, MN) at 4°C overnight.

Techniques: Sequencing

Journal: BMC Cancer

Article Title: Expression of A disintegrin and metalloprotease 8 is associated with cell growth and poor survival in colorectal cancer

doi: 10.1186/1471-2407-14-568

Figure Lengend Snippet: ADAM8 expression status in CRC tumor and normal tissues. A , Expression of ADAM8 in tumor and normal colon tissues detected by IHC. Representative images of IHC staining of colon tissues with anti-ADAM8 antibody on tumor and normal tissues (100× for left panel and 400× for right panel). B , Mean expression levels of ADAM8 mRNA in tumor tissues were significantly higher than that observed in normal tissues ( p = 0.0018). The expression of ADAM8 mRNA was normalized to β-actin mRNA, which served as a control for the input cDNA.

Article Snippet: After deparaffinization in xylene and rehydration through a graded alcohol series, slides were transferred to sodium citrate buffer (Beijing DingguoChangsheng Biotech Co. Ltd, AR-0511) for 15 min in the microware and left at room temperature for 30 min. After blocking endogenous peroxidase, slides were incubated with 10 μg/ml goat polyclonal antibody specific to human ADAM8 (R&D Systems, Inc., Minneapolis, MN) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, Control

Journal: BMC Cancer

Article Title: Expression of A disintegrin and metalloprotease 8 is associated with cell growth and poor survival in colorectal cancer

doi: 10.1186/1471-2407-14-568

Figure Lengend Snippet: Comparison of ADAM8 protein level in normal and tumor tissues by IHC (n = 42)

Article Snippet: After deparaffinization in xylene and rehydration through a graded alcohol series, slides were transferred to sodium citrate buffer (Beijing DingguoChangsheng Biotech Co. Ltd, AR-0511) for 15 min in the microware and left at room temperature for 30 min. After blocking endogenous peroxidase, slides were incubated with 10 μg/ml goat polyclonal antibody specific to human ADAM8 (R&D Systems, Inc., Minneapolis, MN) at 4°C overnight.

Techniques: Comparison, Expressing

Journal: BMC Cancer

Article Title: Expression of A disintegrin and metalloprotease 8 is associated with cell growth and poor survival in colorectal cancer

doi: 10.1186/1471-2407-14-568

Figure Lengend Snippet: Expression of ADAM8 in CRC cell lines. A , Expression of ADAM8 mRNA (top) and protein level (bottom) in HCT8, HT29, SW620, SW480, DLD1, HCT116, LOVO and CACO2 cell lines. B , C , Significantly decreased expression of ADAM8 mRNA (top) and protein levels (bottom) in ADAM8 siRNA-transfected HT29 cells (B) and SW480 cells (C) compared with the control group ( p < 0.001). The expression of ADAM8 mRNA was normalized to β-actin mRNA, which served as the control. Data are expressed as the mean ± standard deviation (SD).

Article Snippet: After deparaffinization in xylene and rehydration through a graded alcohol series, slides were transferred to sodium citrate buffer (Beijing DingguoChangsheng Biotech Co. Ltd, AR-0511) for 15 min in the microware and left at room temperature for 30 min. After blocking endogenous peroxidase, slides were incubated with 10 μg/ml goat polyclonal antibody specific to human ADAM8 (R&D Systems, Inc., Minneapolis, MN) at 4°C overnight.

Techniques: Expressing, Transfection, Control, Standard Deviation

Journal: BMC Cancer

Article Title: Expression of A disintegrin and metalloprotease 8 is associated with cell growth and poor survival in colorectal cancer

doi: 10.1186/1471-2407-14-568

Figure Lengend Snippet: ADAM8 influences proliferation and apoptosis of CRC cells. ADAM8 siRNA-mediated reduction of ADAM8 significantly inhibited proliferation (A) and growth (B and C) of HT29 and SW480 cells compared with the control group. D , Knockdown of ADAM8 significantly induced cell apoptosis in HT29 and SW480 cells compared with controls.

Article Snippet: After deparaffinization in xylene and rehydration through a graded alcohol series, slides were transferred to sodium citrate buffer (Beijing DingguoChangsheng Biotech Co. Ltd, AR-0511) for 15 min in the microware and left at room temperature for 30 min. After blocking endogenous peroxidase, slides were incubated with 10 μg/ml goat polyclonal antibody specific to human ADAM8 (R&D Systems, Inc., Minneapolis, MN) at 4°C overnight.

Techniques: Control, Knockdown

Journal: BMC Cancer

Article Title: Expression of A disintegrin and metalloprotease 8 is associated with cell growth and poor survival in colorectal cancer

doi: 10.1186/1471-2407-14-568

Figure Lengend Snippet: Association between ADAM8 expression in CRC and clinicopathologic characteristics (n = 342)

Article Snippet: After deparaffinization in xylene and rehydration through a graded alcohol series, slides were transferred to sodium citrate buffer (Beijing DingguoChangsheng Biotech Co. Ltd, AR-0511) for 15 min in the microware and left at room temperature for 30 min. After blocking endogenous peroxidase, slides were incubated with 10 μg/ml goat polyclonal antibody specific to human ADAM8 (R&D Systems, Inc., Minneapolis, MN) at 4°C overnight.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Expression of A disintegrin and metalloprotease 8 is associated with cell growth and poor survival in colorectal cancer

doi: 10.1186/1471-2407-14-568

Figure Lengend Snippet: Survival of CRC patients according to the expression status of ADAM8 protein. A , CRC patients with positive ADAM8 had poorer OS than those with negative ADAM8 ( p = 0.037). B , CRC patients with positive ADAM8 had poorer DFS than those with negative ADAM8 ( p = 0.014).

Article Snippet: After deparaffinization in xylene and rehydration through a graded alcohol series, slides were transferred to sodium citrate buffer (Beijing DingguoChangsheng Biotech Co. Ltd, AR-0511) for 15 min in the microware and left at room temperature for 30 min. After blocking endogenous peroxidase, slides were incubated with 10 μg/ml goat polyclonal antibody specific to human ADAM8 (R&D Systems, Inc., Minneapolis, MN) at 4°C overnight.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Expression of A disintegrin and metalloprotease 8 is associated with cell growth and poor survival in colorectal cancer

doi: 10.1186/1471-2407-14-568

Figure Lengend Snippet: Univariate and multivariate analyses of the prognostic factors for 5-year OS of CRC patients (n = 342)

Article Snippet: After deparaffinization in xylene and rehydration through a graded alcohol series, slides were transferred to sodium citrate buffer (Beijing DingguoChangsheng Biotech Co. Ltd, AR-0511) for 15 min in the microware and left at room temperature for 30 min. After blocking endogenous peroxidase, slides were incubated with 10 μg/ml goat polyclonal antibody specific to human ADAM8 (R&D Systems, Inc., Minneapolis, MN) at 4°C overnight.

Techniques:

Journal: BMC Cancer

Article Title: Expression of A disintegrin and metalloprotease 8 is associated with cell growth and poor survival in colorectal cancer

doi: 10.1186/1471-2407-14-568

Figure Lengend Snippet: Five-year OS of patients with positive ADAM8 versus negative ADAM8 tumors in (A) colon cancer ( p = 0.006), (B) T3/4 depth of invasion ( p = 0.023) and (C) N0 stage ( p = 0.032).

Article Snippet: After deparaffinization in xylene and rehydration through a graded alcohol series, slides were transferred to sodium citrate buffer (Beijing DingguoChangsheng Biotech Co. Ltd, AR-0511) for 15 min in the microware and left at room temperature for 30 min. After blocking endogenous peroxidase, slides were incubated with 10 μg/ml goat polyclonal antibody specific to human ADAM8 (R&D Systems, Inc., Minneapolis, MN) at 4°C overnight.

Techniques:

Journal: BMC Cancer

Article Title: Expression of A disintegrin and metalloprotease 8 is associated with cell growth and poor survival in colorectal cancer

doi: 10.1186/1471-2407-14-568

Figure Lengend Snippet: Univariate and multivariate analyses of the prognostic factors for 5-year DFS of CRC patients (n = 292)

Article Snippet: After deparaffinization in xylene and rehydration through a graded alcohol series, slides were transferred to sodium citrate buffer (Beijing DingguoChangsheng Biotech Co. Ltd, AR-0511) for 15 min in the microware and left at room temperature for 30 min. After blocking endogenous peroxidase, slides were incubated with 10 μg/ml goat polyclonal antibody specific to human ADAM8 (R&D Systems, Inc., Minneapolis, MN) at 4°C overnight.

Techniques:

Journal: BMC Cancer

Article Title: Expression of A disintegrin and metalloprotease 8 is associated with cell growth and poor survival in colorectal cancer

doi: 10.1186/1471-2407-14-568

Figure Lengend Snippet: Five-year DFS of patients with positive ADAM8 versus negative ADAM8 tumors in (A) colon cancer ( p = 0.001), (B) T3/4 stage ( p = 0.009), (C) N0 stage ( p = 0.032), (D) TNM II stage ( p = 0.045), (E) adenocarcinoma ( p = 0.027), (F) moderate differentiation ( p = 0.043) and (G) male CRC patients ( p = 0.030).

Article Snippet: After deparaffinization in xylene and rehydration through a graded alcohol series, slides were transferred to sodium citrate buffer (Beijing DingguoChangsheng Biotech Co. Ltd, AR-0511) for 15 min in the microware and left at room temperature for 30 min. After blocking endogenous peroxidase, slides were incubated with 10 μg/ml goat polyclonal antibody specific to human ADAM8 (R&D Systems, Inc., Minneapolis, MN) at 4°C overnight.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Inhibition of HIV-1 release by ADAM metalloproteinase inhibitors

doi: 10.3389/fmicb.2024.1385775

Figure Lengend Snippet: Inhibition of CD62L shedding as detected by ELISA assays. (A,B) CD62L shedding and TNFα release from either PHA or anti-CD3 stimulated CD4 T cells. Both soluble CD62L and TNFα released to supernatant were detected by ELISA (R&D systems). Blank, NT and control designate no cells, no treatment, and isotype control treatment, respectively. (C–F) Anti-CD3 stimulated (C–E) or unstimulated (F) PBMC were treated with indicated inhibitor compounds for 6 (C,E) or 24 (D,F) hours, respectively. Effect of serine-, cysteine-, aspartyl-protease inhibitors (C) , MMP inhibitors (D) as well as titration of MMP inhibitors (E) to the shedding of CD62L from stimulated PBMC. (F) Shedding of CD62L from unstimulated PBMC (F) in the presence of titrating amount of indicated ADAM inhibitors. All statistical analysis were performed using student’s t-test between DMSO and inhibitor treated experiments. Unless indicated with p -values, most treatment did not result in significant differences in the shedding of CD62L. P -values are * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Article Snippet: Western blot human anti-ADAM antibodies were purchased from R&D systems (R&D systems, Inc., Minneapolis, MN) as follows: anti-ADAM8 (MAB1031 and MAB10311), anti-ADAM10 (MAB1427), anti-ADAM15 (MAB935), anti-ADAM 17 (MAB9301), and anti-ADAM19 (AF5050).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Control, Titration

Journal: Clinical Cancer Research

Article Title: ADAM8 as a Novel Serological and Histochemical Marker for Lung Cancer

doi: 10.1158/1078-0432.ccr-04-1436

Figure Lengend Snippet: Fig. 1 Validation of ADAM8 expression in lung tumors and cell lines. A, expression of ADAM8 in a normal lung tissue and 10 clinical NSCLC samples (top panel) and 23 lung cancer cell lines (bottom panel) detected by semiquantitative reverse transcription-PCR analysis. ADC, adenocar- cinoma; SCC, squamous cell carcinoma; LCC, large-cell carcinoma; SCLC, small-cell lung cancer. B–E, representative images after immunohisto- chemical staining of lung tissues, with anti-ADAM8 antibody on tumor tissue microarrays (100). Examples are shown of strong (B), weak (C), and absent (D) ADAM8 expression in ADCs and of no expression in normal lung (E).

Article Snippet: Briefly, 50 g/mL goat polyclonal antihuman ADAM8 antibody (R&D Systems, Inc., Minneapolis, MN) were added after blocking of endogenous peroxidase and proteins, and the sections were incubated with horseradish peroxidase-labeled antigoat IgG (Histofine Simple Stain MAX PO (G), Nichirei, Tokyo, Japan) as the secondary antibody.

Techniques: Biomarker Discovery, Expressing, Reverse Transcription, Staining

Journal: Clinical Cancer Research

Article Title: ADAM8 as a Novel Serological and Histochemical Marker for Lung Cancer

doi: 10.1158/1078-0432.ccr-04-1436

Figure Lengend Snippet: Fig. 2 Cellular localization and secretion of ADAM8 in lung cancer cells. A, expression of ADAM8 on cell surfaces in lung cancer lines NCI-H1373, SK-MES-1, and SBC-5 evaluated by flow cytometric analysis. Mean fluorescence intensity (MFI) was calculated as relative signal intensity values of cells treated with antihuman ADAM8 antibody (green)/cells treated with mouse IgG (control; black). B, standard curve of sandwich ELISA with anti-ADAM8 antibody (X axis, ADAM8 concentration; Y axis, absorbance). C, specific detection of ADAM8 with ELISA in conditioned medium from ADAM8-expressing NCI-H1373 and SK-MES-1 cells and nonexpressing SBC-5 cells. The X axis indicates the relative ELISA values (concentration of secreted ADAM8 from NCI-H1373 or SK-MES-1/concentration of secreted ADAM8 from SBC-5 cells).

Article Snippet: Briefly, 50 g/mL goat polyclonal antihuman ADAM8 antibody (R&D Systems, Inc., Minneapolis, MN) were added after blocking of endogenous peroxidase and proteins, and the sections were incubated with horseradish peroxidase-labeled antigoat IgG (Histofine Simple Stain MAX PO (G), Nichirei, Tokyo, Japan) as the secondary antibody.

Techniques: Expressing, Fluorescence, Control, Sandwich ELISA, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Clinical Cancer Research

Article Title: ADAM8 as a Novel Serological and Histochemical Marker for Lung Cancer

doi: 10.1158/1078-0432.ccr-04-1436

Figure Lengend Snippet: Fig. 3 Serologic concentration of ADAM8 determined by ELISA in patients with lung cancers and in healthy controls. A, distribution of ADAM8 in sera from patients with lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC), or SCLC. Averaged serum levels are shown as black lines. Differences were significant between ADC patients and healthy individuals (P 0.001, Mann-Whitney U test), between SCC patients and healthy individuals (P 0.001) and between SCLC patients and healthy individuals (P 0.001). B, distribution of ADAM8 in sera from patients at various clinical stages of lung cancers. LD indicates limited disease; ED, extensive disease. C, receiver-operating characteristic curve analysis of ADAM8 (blue) and CEA (red) as serum markers for lung cancer (X axis, 1-specificity; Y axis, sensitivity). D, relationship between serum levels of ADAM8 and CEA (X axis, CEA concentration; Y axis, ADAM8 concentration).

Article Snippet: Briefly, 50 g/mL goat polyclonal antihuman ADAM8 antibody (R&D Systems, Inc., Minneapolis, MN) were added after blocking of endogenous peroxidase and proteins, and the sections were incubated with horseradish peroxidase-labeled antigoat IgG (Histofine Simple Stain MAX PO (G), Nichirei, Tokyo, Japan) as the secondary antibody.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Clinical Cancer Research

Article Title: ADAM8 as a Novel Serological and Histochemical Marker for Lung Cancer

doi: 10.1158/1078-0432.ccr-04-1436

Figure Lengend Snippet: Fig. 4 Promotion of invasiveness of mammalian cells transfected with ADAM8-expressing plasmids. A, tran- sient expression of ADAM8 in NIH3T3 and COS-7 cells, detected by semiquan- titative reverse transcription-PCR analy- sis. B, assay demonstrating the invasive nature of NIH3T3 and COS-7 cells in Matrigel matrix after transfection with expression plasmids for human ADAM8. Left panel shows Giemsa staining (200), and right panel represents the relative number of cells migrating through the Matrigel-coated filters (cells with ADAM8-expressing plasmids/cells with mock plasmids). Assays were per- formed three times and in triplicate wells.

Article Snippet: Briefly, 50 g/mL goat polyclonal antihuman ADAM8 antibody (R&D Systems, Inc., Minneapolis, MN) were added after blocking of endogenous peroxidase and proteins, and the sections were incubated with horseradish peroxidase-labeled antigoat IgG (Histofine Simple Stain MAX PO (G), Nichirei, Tokyo, Japan) as the secondary antibody.

Techniques: Transfection, Expressing, Reverse Transcription, Staining

Journal: JCI Insight

Article Title: ADAM8 signaling drives neutrophil migration and ARDS severity

doi: 10.1172/jci.insight.149870

Figure Lengend Snippet: ( A ) Schema for cremaster IVM after i.sc. TNF-α. ( B ) Number of cells adherent to endothelium, ( C ) rolling velocities, and ( D ) quantification of transmigrated Adam8 −/− and Adam8 +/+ neutrophils (right). Representative fields showing leukocyte extravasation (left); arrow indicates direction of emigration. Scale bar, 25 μm. Data for B – D are shown as mean ± SD. **, P < 0.005; Student’s t test. ( E ) Schema for ADAM8 inhibitor experiments after i.t. LPS. ( F ) BAL neutrophils and cytospins (left); scale bar, 25 μm. ( G ) BAL MPO ELISA. ( H ) Quantification of lung tissue and peripheral blood neutrophils. Data for F – H are mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; 2-way ANOVA followed by Holm-Šídák multiple-comparison test. ( I ) Immunostaining of neutrophils (S100A8, green) in 100 μm lung sections. *, large airways. Scale bar, 2 mm. ( J ) Quantification and ( K ) representative images of intravascular (red) and total (green) neutrophils in lung sections at baseline and after i.t. LPS + BK1361 or CP. Ly6G antibody was injected i.v. 10 minutes prior to euthanasia to label intravascular neutrophils; total lung neutrophils were visualized by S100A8 staining (green) and nuclei by DAPI (blue). Neutrophils were quantified from 7 fields/animal ( n = 5). Data in J are mean ± SD, 2-way ANOVA followed by Holm-Šídák multiple comparison test; LPS+CP vs. LPS+BK1361 ( P = 0.0025); LPS+CP vs. PBS ( P = 0.008); LPS+BK1361 vs. PBS (NS). Scale bar, 100 μm ( K ). ( L ) Two-photon lung IVM of MRP8-Cre mTmG mice + BK1361 or CP 3 hours after LPS challenge. Representative images of neutrophil influx (MRP8 + , green). Arrowheads, neutrophil cluster. Scale bar, 50 μm. ( M ) Quantification of neutrophils circulating (track duration < 60 s, left graph) and migrating (track duration > 60 s, right graph) through the lung, n = 3. Blue, baseline; gray, LPS+BK1361; black, LPS+CP. **, P < 0.01; 2-way ANOVA. i.sc., intrascrotal injection.

Article Snippet: Accordingly, human neutrophils were isolated from peripheral blood and preincubated for 2 hours with ADAM8 inhibitors and respective controls (BK-1361 and CP; 1 μg/mL, MAB1031 [R&D Systems] and IgG control; 10 μg/mL) before seeding into matrigel and exposure to an IL-8 gradient (1 μg/mL) on Ibidi Chemotaxis μ-Slides as described above.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Immunostaining, Injection, Staining

Journal: JCI Insight

Article Title: ADAM8 signaling drives neutrophil migration and ARDS severity

doi: 10.1172/jci.insight.149870

Figure Lengend Snippet: ( A ) Domain structure of ADAM8 and Myo1f as interaction partner identified by a yeast-2-hybrid screen using a leukocyte library. ADAM8 CD (aa 678–824) was used as prey and the reporter gene indicated interaction (“+”) via the SH3 domain of Myo1f (clone BP19, aa 1032–1098). ( B ) Co-IP of activated human PMN (hPMN) and ( C ) HL-60 cells using polyclonal ADAM8 (A8) antibody coupled to magnetic beads. Negative controls, beads bound to control IgG (IgG); unconjugated beads (beads). ( D ) Immunofluorescence staining against Myo1f (green) and ADAM8 (red) of hPMN and HL-60 cells. Overlay (yellow) and colocalized pixel map (Coloc Map) indicate colocalization in cell protrusions. Scale bar, 10 μm. ( E ) Co-IP of hPMNs + BK-1361 or CP during activation. ( F ) Relative pixel intensity of Myo1f coprecipitated with ADAM8 normalized to input using ImageJ software (NIH). *, P < 0.05, Student’s t test. ( G ) Colocalization map of hPMNs + BK-1361 or CP as described in D . Scale bar, 10 μm. ( H ) Effect of BK-1361 or SH3 domain blockade (SH3-Inh) on fMLP-induced transendothelial migration of hPMNs ( n = 4 donors). ( I ) Migration across a 3D gel matrix, respectively ( n = 3 donors). ( H and I ) Data are mean ± SD, **, P < 0.01; 2-way ANOVA followed by Holm-Šídák multiple-comparison test. ( J ) 3D chemotactic migration of hPMNs preincubated with BK-1361 or CP toward an IL-8 (1 μg/mL) gradient using chemotaxis μ-Slides. Representative trajectory plots. Triangles indicate orientation of the gradient. Violin plots of ( K ) migration velocity and ( L ) mean Euclidian and ( M ) accumulated distance; BK-1361 (white), SH3 inhibitor (gray), control (black). ( K – M ) Three independent experiments, 30 cells per experiment were analyzed. *, P < 0.05; **, P < 0.01; ***, P < 0.001; 2-way ANOVA followed by Holm-Šídák multiple-comparison test. fMLP, N -formyl-methionyl-leucyl-phenylalanine.

Article Snippet: Accordingly, human neutrophils were isolated from peripheral blood and preincubated for 2 hours with ADAM8 inhibitors and respective controls (BK-1361 and CP; 1 μg/mL, MAB1031 [R&D Systems] and IgG control; 10 μg/mL) before seeding into matrigel and exposure to an IL-8 gradient (1 μg/mL) on Ibidi Chemotaxis μ-Slides as described above.

Techniques: Co-Immunoprecipitation Assay, Magnetic Beads, Control, Immunofluorescence, Staining, Activation Assay, Software, Migration, Comparison, Chemotaxis Assay

Journal: JCI Insight

Article Title: ADAM8 signaling drives neutrophil migration and ARDS severity

doi: 10.1172/jci.insight.149870

Figure Lengend Snippet: ( A ) Adam8 –/– (white bars) or littermate controls ( Adam8 +/+ , black bars) were intranasally instilled with P . aeruginosa (strain PA103) or vehicle control (PBS), then sacrificed 12 hours after infection. ( B ) Mouse Clinical Score for Sepsis (M-CASS) and ( C ) temperature scoring were determined in Adam8 –/– (white) and Adam8 +/+ (black) mice for P . aeruginosa –infected and control animals. ( D ) BAL neutrophils and ( E ) MPO levels. ( F ) WBCs and neutrophils in the peripheral blood of Adam8 +/+ and Adam8 –/– mice infected with P . aeruginosa (blue) or instilled with PBS (gray). ( G ) Representative histologic tissue sections of Adam8 +/+ and Adam8 –/– mice after 12 hours’ P . aeruginosa challenge. Arrowheads, neutrophils in the airspaces; scale bar, 50 μm. (See enlarged sections in .) ( H ) BAL TNF-α and ( I ) total protein. CFU in ( J ) BAL, ( K ) blood, and ( L ) spleen, respectively. ( A and B ) Box-and-whisker plots; ( C – L ) mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, 2-way ANOVA followed by Holm-Šídák multiple-comparison test. ( M ) C57BL/6J mice were inoculated with P . aeruginosa and treated with either cyclic ADAM8 inhibitor peptide (BK-1361, dotted white bars) or control peptide (CP, dotted black bars) at 2 and 12 hours after infection, and sacrificed at 24 hours. ( N ) M-CASS and ( O ) temperature scoring. ( P ) BAL neutrophils and ( Q ) total protein. ( R ) Representative histologic tissue sections of mice treated with CP or BK-1361 during P . aeruginosa challenge. Arrowheads, neutrophils in the airspaces; scale bar, 50 μm. (See enlarged sections in .) CFU in ( S ) BAL, ( T ) blood, and ( U ) spleen. ( N and O ) Box-and-whisker plots; ( Q – U ) mean ± SD; individual data points for each animal are plotted as gray dots. * P < 0.05; ** P < 0.01; Student’s t test.

Article Snippet: Accordingly, human neutrophils were isolated from peripheral blood and preincubated for 2 hours with ADAM8 inhibitors and respective controls (BK-1361 and CP; 1 μg/mL, MAB1031 [R&D Systems] and IgG control; 10 μg/mL) before seeding into matrigel and exposure to an IL-8 gradient (1 μg/mL) on Ibidi Chemotaxis μ-Slides as described above.

Techniques: Control, Infection, Whisker Assay, Comparison

Journal: JCI Insight

Article Title: ADAM8 signaling drives neutrophil migration and ARDS severity

doi: 10.1172/jci.insight.149870

Figure Lengend Snippet: ( A ) Workflow for protease profiling of PMNs at baseline and during activation with TNF-α (20 ng/mL) using 7 FRET-based peptide substrates (PEPDab 005, 008, 010, 011, 013, 014, and 052). To deduce a profile of specific MMP and ADAM activities, peptide cleavage patterns from 3 independent donors were calculated using a nonlinear kinetic model; proteolytic signatures were compared to a reference matrix of catalytic efficiencies to deconvolute protease identities. ( B ) Proteolytic profiling infers increased activity of known neutrophil MMPs and significant levels of active ADAM8; *, P < 0.05; ****, P < 0.0001; 2-way ANOVA followed by Holm-Šídák multiple-comparison test. ( C ) Representative images of ADAM8-positive leukocytes (arrowheads, brown color) in lung tissue of patients with ARDS and healthy controls. Scale bar, 50 μm. (See enlarged sections in .) ( D ) Representative time-lapse fluorimetry of healthy control BAL (gray) and BAL from patients with ARDS from pneumonia (black; ARDS survivor, red; ARDS nonsurvivor) using the most ADAM8-specific FRET reporter, PEPDab013 (mean ± SD of 3 technical replicates); right, box-and-whisker plots of inferred ADAM8 activity in BAL samples of control patients (gray, n = 4), ARDS survivors (black, n = 5), and ARDS nonsurvivors (red, n = 6). *, P < 0.05; ***, P < 0.001; 1-way ANOVA followed by Tukey’s multiple-comparison test. ( E ) Representative time-lapse fluorimetry of BAL of patients with mild (cyan) or severe PGD (blue) using PEPDab 013 (mean ± SD of 3 technical replicates); right, box-and-whisker plots of inferred ADAM8 activity in BAL samples of mild PGD (cyan, n = 16) and severe PGD (blue, n = 16). **, P < 0.005, Student’s t test with Welch’s correction. nd, not detectable.

Article Snippet: Accordingly, human neutrophils were isolated from peripheral blood and preincubated for 2 hours with ADAM8 inhibitors and respective controls (BK-1361 and CP; 1 μg/mL, MAB1031 [R&D Systems] and IgG control; 10 μg/mL) before seeding into matrigel and exposure to an IL-8 gradient (1 μg/mL) on Ibidi Chemotaxis μ-Slides as described above.

Techniques: Activation Assay, Activity Assay, Comparison, Control, Whisker Assay

Journal: JCI Insight

Article Title: ADAM8 signaling drives neutrophil migration and ARDS severity

doi: 10.1172/jci.insight.149870

Figure Lengend Snippet: Neutrophil transmigration requires degradation of ECM and adhesion molecules, as well as cytoskeletal rearrangements. Active ADAM8 has been shown to process membrane proteins with immunological functions (red, ectodomain shedding) and cleave ECM components (blue, ECM degradation) previously ( , , ). Our data suggest a potentially novel contribution of ADAM8 in modulating neutrophil motility by linking its cytoplasmic domain to the cytoskeletal motor protein Myo1f via SH3 domains (red box, cytoskeletal dynamics).

Article Snippet: Accordingly, human neutrophils were isolated from peripheral blood and preincubated for 2 hours with ADAM8 inhibitors and respective controls (BK-1361 and CP; 1 μg/mL, MAB1031 [R&D Systems] and IgG control; 10 μg/mL) before seeding into matrigel and exposure to an IL-8 gradient (1 μg/mL) on Ibidi Chemotaxis μ-Slides as described above.

Techniques: Transmigration Assay, Membrane