Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 1 | Genome-wide screen identifies PACS-2 as an ADAM17 regulator. (a) Cellular model system used for the genome-wide siRNA screen. A total of 21,121 individual genes were knocked down using siRNAs and the effects on ADAM17-mediated shedding of AP-HB-EGF were measured by quantifying AP cell-surface staining after PMA stimulation. (b, c) siRNA-transfected AP-HB-EGF HT1080 (b) or AP-HB-EGF HeLa (c) cells were PMA-stimulated, and the cell medium was analysed for AP activity. The fold change in AP-HB-EGF release was calculated by setting the unstimulated negative control for each experiment to 1, then normalizing the other raw data to this value, and finally calculating the average of all individual experiments. Data in b were compiled from six individual experiments and in c from three individual experiments, each performed in triplicate. (d) AP-HB-EGF MCF-7 cells were siRNA- transfected and stimulated with PMA (left-hand panel) or ionomycin (right-hand panel). Conditioned medium was analysed for AP activity as in b and c. Data were compiled from four individual experiments, each performed in triplicate. In all cases, knockdown was confirmed by western blot. On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by analysis of variance. **Po0.01, ***Po0.001, ****Po0.0001.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Genome Wide, Staining, Transfection, Activity Assay, Negative Control, Knockdown, Western Blot

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 2 | PACS-2 regulates ADAM17-mediated shedding. (a) AP-HB-EGF was transiently expressed in control and Pacs2 / MEFs. Cells were PMA-stimulated, and the cell medium was analysed for AP activity. To overcome differences in transfection efficiency, the fraction of shed AP-HB-EGF was calculated as AP-HB-EGF released to the medium divided by the total amount of AP-HB-EGF in the medium and cell lysate. The fold change in AP-HB-EGF release was then calculated by setting the unstimulated negative control for each experiment to 1, normalizing the other raw data to this value and finally calculating the average of all individual experiments. The PMA-stimulated fold increase in AP-HB-EGF shedding was depicted for each cell line. Data were compiled from four individual experiments, each performed in triplicate. (b) AP-HB-EGF was expressed in Pacs2 / MEFs together with green fluorescent protein (GFP) or PACS-2-HA and shedding analysed as in a. Data were compiled from three individual experiments, each performed in triplicate. (c–e) siRNA-transfected MDA-MB-231 cells were stimulated with PMA (c,d) or TNF-a (e). The medium was analysed by ELISA for shed HB-EGF (c) or TGF-a (d,e), shown as concentrations in pg ml 1. In all cases, data were compiled from three individual experiments, each performed in triplicate. (f) Representative western blots showing knockdown in cells used for ELISA. On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by analysis of variance or unpaired two-tailed Student’s t-test, as appropriate. *Po0.05, **Po0.01, ***Po0.001, ****Po0.0001.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Control, Activity Assay, Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, Western Blot, Knockdown, Two Tailed Test

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 3 | PACS-2 regulates cell-surface availability of mature ADAM17. (a,b) MDA-MB-231 cells were siRNA-transfected and PMA-stimulated. Cells were surface biotinylated and analysed by western blot. ADAM17 levels were normalized to input actin and fold changes were calculated by setting the unstimulated negative control for each experiment to 1, then normalizing the other raw data to this value and finally calculating the average of all individual experiments. Cell-surface levels of mature ADAM17 (a) and total cellular levels of pro and mature ADAM17 (b) are shown with data compiled from four individual experiments. (c,d) Unstimulated cell-surface levels of mature ADAM17 (c) and total cellular levels of pro and mature ADAM17 (d) in control and Pacs2 / MEFs were analysed as in a and b. Data were compiled from four individual experiments in c and three individual experiments in d. (e) ADAM17 was immunoprecipitated from unstimulated control and Pacs2 / MEFs and its activity assessed using an ADAM17 quenched fluorescence peptide substrate. The graph shows the average gradient (fluorescence units/time) of the linear regressions describing enzymatic activity. The data were compiled from three individual experiments, each performed in triplicate. (f) Lysates from control and Pacs2 / MEFs were treated with EndoH or PNGase F and the effects on ADAM17 examined by western blot. The images shown are derived from the same blot but different exposures (indicated by separate panels). The blot shown is representative of three individual experiments. On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by analysis of variance or unpaired two-tailed Student’s t-test, as appropriate. **Po0.01, ***Po0.001, ****Po0.0001.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Transfection, Western Blot, Negative Control, Control, Immunoprecipitation, Activity Assay, Derivative Assay, Two Tailed Test

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 4 | PACS-2 interacts with ADAM17 in endocytic compartments. (a,b) ADAM17 (a) or PACS-2 (b) was immunoprecipitated (IP) from unstimulated and PMA-stimulated MDA-MB-231 cells, and co-immunoprecipitation (co-IP) of PACS-2 (a) or ADAM17 (b) detected by western blot. The blots shown are representative of three individual experiments and # denotes a nonspecific band. (c) The effect of PMA stimulation on PACS-2/ADAM17 co-immunoprecipitation in a and b was quantified. The amount of co-immunoprecipitated PACS-2 (a) or mature ADAM17 (b) was normalized to the amount of immunoprecipitated mature ADAM17 (a) or PACS-2 (b). The unstimulated negative control for each experiment was then set to 1, the other raw data were normalized to this value and finally the average of all individual experiments was calculated. Data were analysed by unpaired two-tailed Student’s t-test. (d) MDA-MB-231 cells were transfected with siRNA and subjected to PLA with or without PMA stimulation. The experiment was performed 10 times in duplicate. Two experiments included the knockdown controls. Scale bar, 7 mm. (e) Before PLA, MDA-MB-231 cells were left unstimulated or treated with PMA and subsequently allowed to internalize fluorescent transferrin for 5 min. Scale bar, 14 mm. Fluorescence intensities were quantified along the lines on the enlarged images and depicted on the graphs. The experiment was performed four times in duplicate.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Negative Control, Two Tailed Test, Transfection, Knockdown, Fluorescence

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 5 | PACS-2 diverts endocytosed ADAM17 away from degradative pathways. (a) MDA-MB-231 cells were surface labelled using cleavable biotin. Labelled cell-surface proteins were allowed to internalize for 30 min and remaining biotinylated proteins on the cell surface were removed (Intern.). Internalized proteins were then allowed to recycle for 30 min, after which recycled protein was stripped from the cell surface (Recyc.). The amount of cell-surface ADAM17 was first normalized to input actin. The percentage internalization was calculated relative to total cell-surface ADAM17 levels (Total). Recycling was determined by first subtracting the amount of biotinylated protein left after recycling from the amount of internalized protein, and then calculated as a percentage of internalized protein. To verify proper stripping of cell-surface proteins, a dish kept at 4 C was processed in parallel (Strip). Data were compiled from five individual experiments. (b) MDA-MB-231 cells were surface labelled using non-cleavable biotin, lysed at time zero, or incubated for 4 h at 37 C to allow internalization and degradation of surface proteins. Percentage degradation was calculated by normalizing the amount of cell-surface ADAM17 to input actin, and dividing the amount remaining after 4 h with the amount present at time zero. Data were compiled from three individual experiments. The images shown are derived from the same blot and same exposure, but have been cropped for clarity (indicated by a separation line). On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by unpaired two-tailed Student’s t-test. *Po0.05.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Stripping Membranes, Incubation, Derivative Assay, Two Tailed Test

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 8 | A model of PACS-2 regulation of ADAM17 and EGFR activity. The ADAM17 proform mainly resides in the secretory apparatus, and during its transit to the cell surface, furin-mediated cleavage generates the mature (active) form. We propose that PACS-2 controls ADAM17 cell-surface availability by diverting endocytosed ADAM17 away from degradative pathways and towards sustained ErbB ligand shedding and EGFR activation. PACS-2 is therefore an important regulator of auto, para and juxtacrine ErbB signalling in vivo.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Activity Assay, Activation Assay, In Vivo

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein.

doi: 10.1016/j.bbamcr.2016.11.003

Figure Lengend Snippet: Figure 1: Tspan3 is a new ADAM10 interaction partner. (A) (I) The principle of the Split- Ubiquitin yeast two Hybrid System is depicted (modified from [27]). ADAM10 is fused to the C- terminal part of ubiquitin (Cub) and an artificial transcription factor LexA (LexA-VP16) expressed in yeast together with N-terminal NubG tagged proteins from a murine brain cDNA library. The close proximity between the ADAM10 bait protein and an interaction partner leads to the reconstituion of ubiquitin. Cellular ubiquitin proteases release the transcription factor LexA, which activates HIS3 gene transcription and allows yeast clones to grow on selective media plates lacking histidin (His). (II-III) ADAM10 bait protein was co-expressed with the identified Tspan3 (Tsp3)

Article Snippet: The following antibodies were used: anti-ADAM10 antibody EPR5622 (IB, Abcam, Cambridge, UK previously GenTex, Irvine, USA), anti-ADAM10 antibody raised against a the C-terminus of ADAM10 (IP, [12]), MAB946 anti-ADAM10 ectodomain (FACS, R&D Systems, Minneapolis, USA), anti-ADAM17 antibody A300D [23], anti-actin (IB, Sigma-Aldrich, Munich, Gemany), B63.2 anti-APP C-terminus (IB, a kind gift of Wim Annaert, Leuven, Belgium), anti-EEA1 (IF, Cell Signaling Technology, Danvers, USA), FL-335 anti-GAPDH (Santa Cruz Biotechnology, Dallas, USA), 9B11 anti-myc (IB and IF, Cell Signaling Technology, Danvers, USA), anti-myc (IP, GTX30518, GenetTex, Irvine, USA), anti-N-Cadherin (BD Transduction Laboratories, Heidelberg, Germany), anti-PDI (A6) (Abcam, Cambridge, UK), MAB 5232 anti-Presenilin-1 C-terminal loop (IB, Merck Millipore, Darmstadt, Germany), AB1563 anti-Presenilin-1 N-terminal (IB, Merck Millipore, Darmstadt, Germany), L2T2 anti-LIMP-2 (IF, IB [24]), anti-PDI (IF, Santa Cruz Biotechnology, Heidelberg, Germany), POM1 anti-PrP c (IB, Prof. Dr. A. Aguzzi, Zürich, Switzerland), anti-Rab5 (IF, Synaptic Systems, Göttingen, Germany), H4B4 anti-LAMP2 (IF, DSHB, Iowa Ciy, USA), anti-E7 beta-Tubulin (DSHB Iowa Ciy, USA), anti-Tspan3 (IB, IP, IF, IHC, antibody produced by Pineda, Berlin, Germany against a synthetic peptide corresponding to the last 19 amino acids of the C-terminus of mouse Tspan3).

Techniques: Ubiquitin Proteomics, Modification, cDNA Library Assay, Clone Assay

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein.

doi: 10.1016/j.bbamcr.2016.11.003

Figure Lengend Snippet: Figure 2: Tspan3 expression accelerates ADAM10 CTF generation without increasing ADAM10 surface levels. (A) Murine Tspan3-myc (Tsp3) and Tspan15-myc (Tsp15) expressed in HeLa cells (I). After immunoblotting ADAM10 was detected using an ADAM10-specific C-

Article Snippet: The following antibodies were used: anti-ADAM10 antibody EPR5622 (IB, Abcam, Cambridge, UK previously GenTex, Irvine, USA), anti-ADAM10 antibody raised against a the C-terminus of ADAM10 (IP, [12]), MAB946 anti-ADAM10 ectodomain (FACS, R&D Systems, Minneapolis, USA), anti-ADAM17 antibody A300D [23], anti-actin (IB, Sigma-Aldrich, Munich, Gemany), B63.2 anti-APP C-terminus (IB, a kind gift of Wim Annaert, Leuven, Belgium), anti-EEA1 (IF, Cell Signaling Technology, Danvers, USA), FL-335 anti-GAPDH (Santa Cruz Biotechnology, Dallas, USA), 9B11 anti-myc (IB and IF, Cell Signaling Technology, Danvers, USA), anti-myc (IP, GTX30518, GenetTex, Irvine, USA), anti-N-Cadherin (BD Transduction Laboratories, Heidelberg, Germany), anti-PDI (A6) (Abcam, Cambridge, UK), MAB 5232 anti-Presenilin-1 C-terminal loop (IB, Merck Millipore, Darmstadt, Germany), AB1563 anti-Presenilin-1 N-terminal (IB, Merck Millipore, Darmstadt, Germany), L2T2 anti-LIMP-2 (IF, IB [24]), anti-PDI (IF, Santa Cruz Biotechnology, Heidelberg, Germany), POM1 anti-PrP c (IB, Prof. Dr. A. Aguzzi, Zürich, Switzerland), anti-Rab5 (IF, Synaptic Systems, Göttingen, Germany), H4B4 anti-LAMP2 (IF, DSHB, Iowa Ciy, USA), anti-E7 beta-Tubulin (DSHB Iowa Ciy, USA), anti-Tspan3 (IB, IP, IF, IHC, antibody produced by Pineda, Berlin, Germany against a synthetic peptide corresponding to the last 19 amino acids of the C-terminus of mouse Tspan3).

Techniques: Expressing, Western Blot

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein.

doi: 10.1016/j.bbamcr.2016.11.003

Figure Lengend Snippet: Figure 3: Tspan3 expression leads to an ADAM10-mediated increase of the APP -CTF (C83). (A) Tspan3-myc (Tsp3) expression in HeLa cells increases the levels of the APP-CTFs but does not affect the N-Cadherin CTF production. Expressing Tspan15-myc (Tsp15) only moderately

Article Snippet: The following antibodies were used: anti-ADAM10 antibody EPR5622 (IB, Abcam, Cambridge, UK previously GenTex, Irvine, USA), anti-ADAM10 antibody raised against a the C-terminus of ADAM10 (IP, [12]), MAB946 anti-ADAM10 ectodomain (FACS, R&D Systems, Minneapolis, USA), anti-ADAM17 antibody A300D [23], anti-actin (IB, Sigma-Aldrich, Munich, Gemany), B63.2 anti-APP C-terminus (IB, a kind gift of Wim Annaert, Leuven, Belgium), anti-EEA1 (IF, Cell Signaling Technology, Danvers, USA), FL-335 anti-GAPDH (Santa Cruz Biotechnology, Dallas, USA), 9B11 anti-myc (IB and IF, Cell Signaling Technology, Danvers, USA), anti-myc (IP, GTX30518, GenetTex, Irvine, USA), anti-N-Cadherin (BD Transduction Laboratories, Heidelberg, Germany), anti-PDI (A6) (Abcam, Cambridge, UK), MAB 5232 anti-Presenilin-1 C-terminal loop (IB, Merck Millipore, Darmstadt, Germany), AB1563 anti-Presenilin-1 N-terminal (IB, Merck Millipore, Darmstadt, Germany), L2T2 anti-LIMP-2 (IF, IB [24]), anti-PDI (IF, Santa Cruz Biotechnology, Heidelberg, Germany), POM1 anti-PrP c (IB, Prof. Dr. A. Aguzzi, Zürich, Switzerland), anti-Rab5 (IF, Synaptic Systems, Göttingen, Germany), H4B4 anti-LAMP2 (IF, DSHB, Iowa Ciy, USA), anti-E7 beta-Tubulin (DSHB Iowa Ciy, USA), anti-Tspan3 (IB, IP, IF, IHC, antibody produced by Pineda, Berlin, Germany against a synthetic peptide corresponding to the last 19 amino acids of the C-terminus of mouse Tspan3).

Techniques: Expressing

Journal: Scientific Reports

Article Title: ADAM9 promotes lung cancer progression through vascular remodeling by VEGFA, ANGPT2, and PLAT

doi: 10.1038/s41598-017-15159-1

Figure Lengend Snippet: ADAM9-regulated genes with vascular remodeling functions. ( A ) Ingenuity Pathway Analysis, by comparing gene expression in control versus ADAM9 knockdown cells, showed that the gene network functioned in cell death and survival, cardiovascular disease, and hematological disease. ( B ) Ratio of differential gene expression in microarray analysis (control shGFP Bm7 cells/ ADAM9 knockdown Bm7 cells). ( C – E ) Western blot of ADAM9, ANGPT2, and VEGFA in control and ADAM9 knockdown cells from Bm7 lung cancer cells ( C ), Brmx2 lung cancer cells ( D ), and A549 lung cancer cells ( E ). ( F ) Western blot of ADAM9, VEGFA, and ANGPT2 in Bm7 cells treated with BB94 (a broad spectrum of metalloproteinase inhibitor). ( G ) Western blot of ADAM9, VEGFA, and ANGPT2 in control and ADAM9 knockout Brmx2 cells transiently transfected with plasmids of empty control ( E ), ADAM9 wild-type (WT), or ADAM9 mutant (MT) (E348A). EF1α or GAPDH served as the loading control in ( C – G ). Some cropped blots in ( C – G ) were displayed and the full-length blots were shown in Supplementary Figure .

Article Snippet: The antibodies used in the study were against ADAM9 (MAB939, R&D Systems, Minneapolis, MN, USA), mouse ADAM9 (AF949, R&D Systems), ANGPT2 (AF623, R&D Systems), VEGFA (ABS82, Millipore, Billerica, MA, USA), CD31 (ab28364, Abcam), and elongation factor 1 α (EF1α, #05–235, Millipore).

Techniques: Gene Expression, Control, Knockdown, Microarray, Western Blot, Knock-Out, Transfection, Mutagenesis

Journal: Scientific Reports

Article Title: ADAM9 promotes lung cancer progression through vascular remodeling by VEGFA, ANGPT2, and PLAT

doi: 10.1038/s41598-017-15159-1

Figure Lengend Snippet: ADAM9 promotes angiogenesis through VEGFA. ( A ) Concentrations of VEGFA were measured by ELISA in conditioned media of control and ADAM9 knockdown Bm7 cells. ( B , C ) Tube formation assays of HUVECs were performed by treatment with growth media alone (as control) or the conditioned media of indicated cells. Conditioned media of shADAM9-E Bm7 cells (B) and shADAM9-H Bm7 cells ( C ) were used for tube formation assays. ( D ) Tube formation assays were performed by treating HUVECs with the conditioned media of Bm7-shGFP cells in the presence or absence of 5 μg/ml Avastin pre-incubation (white bars), or with the conditioned media of Bm7-shADAM9 cells in the presence or absence of 100 ng/ml VEGF (black bars). Top, photographs of representative experiments for control, GFP knockdown, and ADAM9 knockdown; bottom, quantitative data for tube formation. Bars represent the mean ± SD of three replicate samples from one representative experiment. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used in the study were against ADAM9 (MAB939, R&D Systems, Minneapolis, MN, USA), mouse ADAM9 (AF949, R&D Systems), ANGPT2 (AF623, R&D Systems), VEGFA (ABS82, Millipore, Billerica, MA, USA), CD31 (ab28364, Abcam), and elongation factor 1 α (EF1α, #05–235, Millipore).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Knockdown, Incubation

Journal: Scientific Reports

Article Title: ADAM9 promotes lung cancer progression through vascular remodeling by VEGFA, ANGPT2, and PLAT

doi: 10.1038/s41598-017-15159-1

Figure Lengend Snippet: ADAM9 disrupts the integrity of brain endothelial cells through ANGPT2 and PLAT. ( A ) VE-cadherin staining of HBMECs treated with concentrated media from the indicated cancer cells. After incubation, cells were stained with anti-VE-cadherin antibody (red) and DAPI (blue). For quantitation, the membrane length stained by anti-VE-cadherin antibody in HBMECs was calculated and normalized to HBMEC nuclear numbers. Anti-ANGPT2 antibody was pre-incubated with concentrated conditioned media before treatment of HBMECs. ( B ) Permeability of HBMECs treated with concentrated conditioned media from the indicated cancer cells. Anti-ANGPT2 antibody was pre-incubated with concentrated conditioned medium before treatment of HBMECs. * P < 0.05. ( C ) VE-cadherin staining of HBMECs treated with the indicated conditioned media or with recombinant tPA protein reveals the integrity of HBMECs.

Article Snippet: The antibodies used in the study were against ADAM9 (MAB939, R&D Systems, Minneapolis, MN, USA), mouse ADAM9 (AF949, R&D Systems), ANGPT2 (AF623, R&D Systems), VEGFA (ABS82, Millipore, Billerica, MA, USA), CD31 (ab28364, Abcam), and elongation factor 1 α (EF1α, #05–235, Millipore).

Techniques: Staining, Incubation, Quantitation Assay, Membrane, Permeability, Recombinant

Journal: Scientific Reports

Article Title: ADAM9 promotes lung cancer progression through vascular remodeling by VEGFA, ANGPT2, and PLAT

doi: 10.1038/s41598-017-15159-1

Figure Lengend Snippet: ADAM9 knockout significantly inhibits tumor growth and metastasis in vivo . ( A ) Western blot of ADAM9 knockout in mouse lung cancer TC1 cells. WT, wild-type; #1 and #2 represent two independent stable clones with ADAM9 knockout. ( B ) Wild-type ( n = 7) or ADAM9 knockout ( n = 7) TC1 cells were injected subcutaneously into C57BL/6 mice. The photograph ( top ) shows subcutaneous tumors removed from each group after sacrifice; the graph ( bottom ) shows the quantification of tumor volume at the indicated days after inoculation. ( C ) Western blot of ADAM9, ANGPT2, and VEGFA from subcutaneous (s.c.) tumors of each group. ( D ) Tumor tissues were processed for IHC staining of CD31. The images were magnified at 100x ( top , scale bar 200 μm). CD31 staining was quantified as the percentage of positively stained density ( bottom ). ( E ) WT ( n = 5) and ADAM9 KO ( n = 5) TC1 cells were injected intravenously via the tail vein of mice. The photograph shows metastatic tumors in the lung from each group ( top ) and the weight of whole lung tumors was measured from the two groups ( bottom ). ( F ) Metastasis in the lung from WT or ADAM9 KO TC1 cells injection intravenously appeared as dense tumor clusters (T) following hematoxylin and eosin staining (H&E). ( G ) Lung tumor tissues were processed for IHC staining of ADAM9. Images were scanned with Aperio software; digital resolution is 0.5 μm per pixel. Scale bar is 500 μm. Vessels were highlighted by red broken line (left, upper). The representative images of ADAM9 staining in tumor with or without vessels were showed (left, lower). The scale bar is 100 μm. The intensities of ADAM9, H score, were quantified by Aperio ImageScope software program (right). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The antibodies used in the study were against ADAM9 (MAB939, R&D Systems, Minneapolis, MN, USA), mouse ADAM9 (AF949, R&D Systems), ANGPT2 (AF623, R&D Systems), VEGFA (ABS82, Millipore, Billerica, MA, USA), CD31 (ab28364, Abcam), and elongation factor 1 α (EF1α, #05–235, Millipore).

Techniques: Knock-Out, In Vivo, Western Blot, Clone Assay, Injection, Immunohistochemistry, Staining, Software

Journal: Scientific Reports

Article Title: ADAM9 promotes lung cancer progression through vascular remodeling by VEGFA, ANGPT2, and PLAT

doi: 10.1038/s41598-017-15159-1

Figure Lengend Snippet: Simultaneous high expression of ADAM9 and VEGFA correlates with poor outcome in lung adenocarcinoma patients. ( A ) Kaplan-Meier survival analyses of lung adenocarcinoma patients in each indicated data set, with patients categorized by ADAM9 and VEGFA gene expression. DH, dual high; SH, single high; DL, dual low. High and low are above and below the median, respectively. ( B ) Cox proportional hazards regression analyses of overall survival in each dataset of lung adenocarcinoma patients, grouped by ADAM9 and VEGFA expression. ( C ) Ranking the P -value of ADAM9 plus VEGFA among all P -values of profile genes by combining a fixed predictor with the other randomly selected genes from the same dataset. Fixed predictor: ADAM9 in the GSE8894 dataset and VEGFA in the Shedden dataset. The P -value of ADAM9 high /VEGFA high versus ADAM9 low /VEGFA low is marked by the red line.

Article Snippet: The antibodies used in the study were against ADAM9 (MAB939, R&D Systems, Minneapolis, MN, USA), mouse ADAM9 (AF949, R&D Systems), ANGPT2 (AF623, R&D Systems), VEGFA (ABS82, Millipore, Billerica, MA, USA), CD31 (ab28364, Abcam), and elongation factor 1 α (EF1α, #05–235, Millipore).

Techniques: Expressing, Gene Expression

Journal: Scientific Reports

Article Title: ADAM9 promotes lung cancer progression through vascular remodeling by VEGFA, ANGPT2, and PLAT

doi: 10.1038/s41598-017-15159-1

Figure Lengend Snippet: Simultaneous high expression of ADAM9 and ANGPT2 correlates with poor outcome in lung adenocarcinoma patients. ( A ) Kaplan-Meier survival analyses of lung adenocarcinoma patients in each indicated data set, with patients categorized by ADAM9 and ANGPT2 gene expression. Labels are defined as in Fig. . ( B ) Cox proportional hazards regression analyses of overall survival in each dataset of lung adenocarcinoma patients, grouped by ADAM9 and ANGPT2 expression. ( C ) Ranking the P -value of ADAM9 plus ANGPT2 among all P -values of profile genes by combining a fixed predictor with the other randomly selected genes from the same dataset. Fixed predictor: ADAM9 in the GSE8894 and GSE11969 datasets and ANGPT2 in the Shedden dataset. The P -value of ADAM9 high /ANGPT2 high versus ADAM9 low /ANGPT2 low is marked by the red line.

Article Snippet: The antibodies used in the study were against ADAM9 (MAB939, R&D Systems, Minneapolis, MN, USA), mouse ADAM9 (AF949, R&D Systems), ANGPT2 (AF623, R&D Systems), VEGFA (ABS82, Millipore, Billerica, MA, USA), CD31 (ab28364, Abcam), and elongation factor 1 α (EF1α, #05–235, Millipore).

Techniques: Expressing, Gene Expression

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Biochemical evidence for ADAM10-mediated CA IX ECD cleavage. (A) Verification of the cleavage activity of rhADAM10 catalytic domain towards the fluorogenic peptide Mca-K-P-L-G-L-Dpa-A-R-NH 2 . The peptide was used at the final concentration of 10 µM in a total of 100 µl reaction mixture with 10 ng of the rhADAM10. The cleavage was allowed to proceed for 30, 40, 50 and 120 min. Time-related increase of the fluorescence emitted from the peptide proves that rhADAM10 was active in comparison to negative control without rhADAM10. (B) ELISA analysis of CA IX ECD in culture medium obtained from CHOwt-FL-CA IX and CHOwt-NS-CA IX cells transiently expressing FL-CA IX and NS-CA IX, respectively. Transfected cells were treated with rhADAM10 (500 ng/ml) for 24 h (Data were analyzed by Student's t-test). (C) Incubation of CHOwt-FL-CA IX cells with ADAM17 activator PMA (20 µM, 3 h), ADAM10 activator IONO (1 µg/ml, 3 h) or rhADAM10 (500 ng/ml, 24 h). Data were analyzed using one-way ANOVA followed by Dunnett's test. Experiments were performed in triplicates and repeated twice. The results were expressed as the mean ± SD. **P<0.01 and ***P<0.001. ADAM, a disintegrin and metalloproteinase; CA IX, carbonic anhydrase IX; rhADAM10, recombinant human ADAM10; ECD, ectodomain; FL, full-length; NS, non-shed; IONO, ionomycin; PMA, phorbol 12-myristate 13-acetate; ns, non-significant.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Activity Assay, Concentration Assay, Fluorescence, Comparison, Negative Control, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Incubation, Recombinant

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Co-localization of ADAM10 and CA IX in C33a-FL-CA IX cells. Immunofluorescence of (A) C33a-FL-CA IX cells expressing CA IX, and (B) control C33a-neo cells using ADAM10-specific antibody. Nuclei were counterstained with DAPI. PLA performed in (C) C33a-FL-CA IX and (D) C33a-neo cells using rabbit anti-human CA IX-specific antibody and mouse anti-human ADAM10 antibody. Red PLA signal indicating the interaction of CA IX with ADAM10 was clearly visible only in C33a cells expressing FL CA IX. Experiment was performed in triplicates and repeated twice. Magnification, ×630. Scale bar, 10 µm. ADAM, a disintegrin and metalloproteinase; CA IX, carbonic anhydrase IX; PLA, proximity ligation assay; FL, full-length.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Immunofluorescence, Expressing, Control, Proximity Ligation Assay

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Localization of ADAM10 (green) in response to CA9hu-1 antibody-induced CA IX internalization (red). C33a-FL-CA IX cells were pre-incubated either with anti-ADAM10 antibody alone (ctrl, A-D) or with anti-ADAM10 antibody together with the internalization-inducing anti-CA IX antibody CA9hu-1 (E-H) 30 min at 4°C. Plasma membrane staining signal for ADAM10 and CA IX was observed at all time points in the absence of CA9hu-1 pre-treatment. On the other hand, CA9hu-1-induced internalization of CA IX as well as ADAM10 was visible after 2 and 4 h at 37°C (E and F), while both molecules showed recycling to plasma membrane after 8 and 24 h. Overlapped staining signals were evident in all samples. Magnification, ×630. Scale bar, 20 µm. Experiment was performed in triplicates and repeated twice. ADAM, a disintegrin and metalloproteinase; CA IX, carbonic anhydrase IX.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Incubation, Clinical Proteomics, Membrane, Staining

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: GI-induced internalization of ADAM10 and targeting of ADAM10 by RNA interference or by a dominant-negative ADAM10 mutant ∆MP. (A-H) C33a-FL-CA IX cells were pre-incubated with anti-ADAM10 antibody at 4°C. (A-D) Cells showed the plasma membrane staining signal for ADAM10 (green) in absence of GI at 37°C. (E-H) GI-induced internalization of ADAM10 was clearly visible as cytoplasmic staining signal in all incubation periods at 37°C. (I) Direct targeting of ADAM10 was performed by RNA interference and (J) expression of a dominant-negative mutant ∆MP with and without GI treatment. Control cells were transfected with either an esiRNA targeting RLUC or an empty vector (pcDNA3.1). Culture media collected from transfected cells incubated in presence and absence of GI for 24 h were collected and subjected to ELISA analysis for detection of CA IX ECD. Experiment was performed in triplicates and repeated six times. Data were analyzed by one-way ANOVA followed by Dunnett's test. Results were expressed as the mean percentage of CA IX ECD shedding with control cells set as 100% ± SD. ***P<0.001. GI, GI254023X; ADAM, a disintegrin and metalloproteinase; ∆MP, dominant-negative mutant of ADAM10; esiRNA, enzymatically-prepared small interfering RNA; CA IX, carbonic anhydrase IX; ECD, ectodomain.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Dominant Negative Mutation, Mutagenesis, Incubation, Clinical Proteomics, Membrane, Staining, Expressing, Control, Transfection, esiRNA, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Small Interfering RNA

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Additive effect of ADAM10 and ADAM17 activation/inhibition. (A) Quantitative PCR analysis of ADAM10 and ADAM17 mRNA levels in C33a-FL-CA IX and C33a-NS-CA IX cells normalized to the level of β-actin mRNA. (B) Western blot analysis of ADAM10, ADAM17 and CA IX protein in C33a-FL-CA IX and C33a-NS-CA IX cells. The anti-actin antibody was used as a loading control. (C and D) ELISA analysis of CA IX ECD in culture medium collected from C33-FL-CA IX cells after the treatment with IONO (1 µg/ml), PMA (20 µM), IONO + PMA (1 µg/ml; 20 µM), D1(A12) antibody (200 nM), GI inhibitor (1 µM) or D1(A12) + GI (200 nM; 1 µM) for 3 h in comparison to non-treated cells (CTRL). Experiment was performed in triplicates and repeated two times. Data were analyzed by (A) Student's t-test and (C and D) one-way ANOVA followed by Dunnett's test. Results were expressed as the mean relative levels of mRNA or CA IX ECD ± SD. ***P<0.001. ADAM, a disintegrin and metalloproteinase; FL, full-length; CA IX, carbonic anhydrase IX; NS, non-shed; ECT, ectodomain; IONO, ionomycin; PMA, phorbol 12-myristate 13-acetate; GI, GI254023X.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Activation Assay, Inhibition, Real-time Polymerase Chain Reaction, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Scheme illustrating CA IX ECD shedding by ADAM10 and ADAM17 proteinases. Picture depicts differences in regulatory components that differentially affect ADAMs biosynthesis and processing at various levels of their expression and activation and thereby can potentially influence CA IX ECD cleavage. Based on ( , – ). CA IX, carbonic anhydrase IX; ADAM, a disintegrin and metalloproteinase; ECD, ectodomain.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Expressing, Activation Assay

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 2 | Influence of metalloproteinases on the expression and release of CXCL16. (a) Analysis of soluble CXCL16 released from primary TALDCs with a CXCL16-specific ELISA. Cells were preincubated with different proteinase inhibitors for 15 min before IFN-g (24 ng/ml) was added for 24 h. Application of IFN-g (24 ng/ml) increased the amount of soluble CXCL16 in the supernatants of TALDCs. The ADAM10-specific metalloproteinase inhibitor, GI254023X (GI), and the broad-spectrum metalloproteinase inhibitors, GM6001 and TAPI-2, reduced the IFN-g-induced CXCL16 release. Data are mean±s.d. (n ¼ 3); ***Po0.001 versus control; ###Po0.001, ##Po0.01; #Po0.05 versus cytokine-treated cells. (b) Inhibition of metalloproteinases increased cellular CXCL16 protein expression in TALDCs. Fifteen minutes before application of IFN-g, cells were pretreated with the indicated concentration of the metalloproteinase inhibitors GI254023X, GM6001, and TAPI-2. CXCL16 protein levels were measured by western blot analysis and b-actin was used as a loading control. (c) Knockdown of CXCL16 with CXCL16-specific siRNA in TALDCs is shown by western blot analysis. (d) Application of pharmacological inhibitors of metalloproteinases increased the amount of cellular CXCL16. Cell lysates were isolated as described under Materials and Methods, and 15 mg total proteins were used to measure the amount of cell-expressed CXCL16 by CXCL16-specific ELISA. Data are mean þ s.d. (n ¼ 5). Statistically significant release of CXCL16 (Po0.001) is indicated by asterisk. (e) Suppression of ADAM10 protein levels by RNA interference in the presence and absence of IFN-g. Cell lysates of TALDCs were prepared 48 h after transfection with siRNA specific for ADAM10 and an unspecific siRNA. Mock-transfected cells treated with the transfection lipid in the absence of siRNA were used as a control. Western blot analysis of ADAM10 protein expression was performed. Blots were reprobed with an antibody specific for b-actin as a loading control. (f) Inhibition of ADAM10 by siRNA reduced the IFN-g-stimulated CXCL16 release in TALDCs. Supernatants of siRNA-treated and IFN-g- stimulated or unstimulated cells were analyzed for soluble CXCL16 using a CXCL16-specific ELISA (n ¼ 3); ***Po0.001 versus control; ###Po0.001 versus IFNg-treated cells.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Inhibition, Concentration Assay, Western Blot, Knockdown, Isolation, Transfection

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 3 | Localization of ADAM10 protein in normal human kidney. (a) In the overview, ADAM10 protein is mainly expressed in tubular cells, but expression was also found in glomerular cells. Serial sections of normal renal tissue represented constitutive ADAM10 expression in aquaporin-2 (e), calbindin D-28K (f), and Tamm–Horsfall glycoprotein (g) expressing tubular profiles, which correspond to CD, (DCT, CNT), and TAL, respectively. Proximal tubules were devoid of any specific ADAM10 staining. (h) Immunofluorescent detection of ADAM10 (red fluorescence) in aquaporin-2-positive principal cells of the CD (green fluorescence). A prominent overlap of both signals in the same cells can be seen, indicated in the examples by asterisks. In contrast, arrows indicate cells negative for either aquaporin-2 or ADAM10, thus representing presumable intercalated cells. (i) ADAM10 is not expressed in the intercalated cells of the CD. Note: arrows represent H þ-ATPase-expressing intercalated cells (green colour) that do not express ADAM10 (red colour); compare with the merged view. (j) Confocal immunofluorescence analysis of ADAM10 and CXCL16 expression in a cortical CD. Tissue section was stained with Alexa Fluor 488 and Cy3 secondary antibodies to visualize the localization of CXCL16 (red) and ADAM10 (green) proteins, respectively. Both signals can be confined to same segment-specific cells; see merged picture on the right.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Staining, Fluorescence, Immunofluorescence

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 4 | Supernatants from ADAM10- and CXCL16 siRNA-transfected TALDCs decreased the chemotaxis of Jurkat T cells. (a) Surface expression of the chemokine receptor CXCR6 in Jurkat T cells analyzed by fluorescence-activated cell sorting analysis. (b) Inhibition of CXCL16 protein expression by siRNA fully abolished release of CXCL16 in TALDCs. Cells were transfected with different siRNA duplexes for inhibition of ADAM10 or CXCL16, an unspecific siRNA (scrambled), or not transfected with siRNA (mock). A total of 24 h after transfection, TALDCs were stimulated with IFN-g for 24 h, where indicated, and supernatants were collected. Soluble CXCL16 was determined by a CXCL16-specific ELISA (n ¼ 4). Data are mean±s.d.; ***Po0.001 versus control; ###Po0.001 versus IFN-g-treated cells. (c) Decrease of soluble CXCL16 correlates with a reduction in the migration of Jurkat T cells. Supernatants shown in Figure 5b were used for chemotaxis assays (n ¼ 2). Recombinant CXCL16 induced migration of CXCR6-expressing Jurkat T cells about threefold; **Po0.01; *Po0.05 versus control.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Transfection, Chemotaxis Assay, Expressing, Fluorescence, FACS, Inhibition, Enzyme-linked Immunosorbent Assay, Control, Migration, Recombinant

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 5 | Increased urinary CXCL16 correlated with focally apical CXCL16 expression in tubular cells of renal allografts with the histopathological diagnosis of ATN. Six allograft biopsies of patients diagnosed with ATN (cases A1–A6; clinical parameters and CXCL16 analysis are shown in Table 1) were analyzed by CXCL16 immunohistochemical analysis (a–f) and with ADAM10 (green colour) and CXCL16 (red colour) double immunofluorescence staining (g–l). Notably, all patients showed focally increased apical CXCL16 expression in renal tubuli. Strongest apical CXCL16, seen in patient A4 (d), correlated with the highest amount of urinary CXCL16 (Table 1). In contrast, ADAM10 expression (green colour) was not significantly changed in ATN patients (A1–A6) compared with normal kidney (g–l). (m) Urinary CXCL16 measured by a CXCL16-specific ELISA in healthy volunteers (normal), in patients with IR, and in patients with ATN. Data are mean±s.d.; **Po0.01 versus control.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Biomarker Discovery, Immunohistochemical staining, Double Immunofluorescence Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 6 | Increased ADAM10 expression in allograft biopsies of kidney transplant patient with the clinical and histopathological diagnosis of acute IR. Renal allograft biopsies of patients diagnosed with IR were analyzed by double immunofluorescence for ADAM10 and CXCL16 expression in comparison with normal kidney (upper panel). Clinical features and ADAM10 analysis of patients are listed in Table 2. Tissue sections were stained with Alexa Fluor 488 and Cy3 secondary antibodies to visualize the localization of CXCL16 (red) and ADAM10 (green) proteins, respectively. In some tubuli of patient B1 (marked with arrows), strong basolateral ADAM10 expression and no CXCL16 expression could be seen (middle panel). In contrast, in few tubuli of the same patient (marked with a star) both molecules colocalized. In the lower panel (patient B5), strong coexpression of ADAM10 and CXCL16 in a tubule is visible, accompanied with interstitial inflammatory infiltrates.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Biomarker Discovery, Immunofluorescence, Comparison, Staining