Journal: Scientific Reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: ADAM10 and ADAM17 are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.

Article Snippet: Anti-N-terminal mouse Robo4 antibody (Abcam, ab10547), anti-N-terminal human Robo4 antibody (R&D Systems, MAB2454), anti-FLAG antibody (Thermo Fisher Scientific, 14-6681-82), anti-HA antibody (Chromotek, #7c9-100), anti-intracellular Robo4 domain antibody (Santa Cruz Biotechnology, sc46497), anti-ADAM10-pro antibody (Abcam, ab39178), anti-ADAM10 antibody (Bioss, bs-3574R; LSbio, C497146-200; Novus, #NBP1-76973), anti-ADAM17 antibody (Bioss, 4236R; Novus #NBP2-61719), anti-Synecan-1 antibody (Santa Cruz Biotechnology, sc-5632), anti-actin antibody (Sigma Aldrich, A2228), anti-GAPDH antibody (R&D Systems, AF5718), polyclonal anti-Unc5B antibody (R&D Systems, AF1006), anti-N-terminal CDH5 (Thermo Fisher Scientific, 14-1441-82), anti-CD31 (BD, #550274), HRP-conjugated anti-His antibody (Alpha Diagnostic International, HISP12-HRP), HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, sc-2030), HRP-conjugated donkey anti-goat IgG antibody (Santa Cruz Biotechnology, sc-2020), and HRP-conjugated goat anti-mouse IgG antibody (Invitrogen, 62-6520).

Techniques: Expressing, Quantitative RT-PCR, Inhibition, Flow Cytometry, Staining, Transfection, shRNA, Western Blot

Journal: Scientific Reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: Inhibition of ADAM10 and ADAM17 increases Robo4 C-terminal Fragment, and Robo4 co-localizes with ADAM10 and ADAM17 in endothelial cells. ( A ) Human Robo4-HA-FLAG expression. The expression of hRobo4-HA-FLAG in dEC lysate was probed with an anti-human Robo4 ectodomain antibody in Western blot. A 130 KDa band was detected. ( B ) Pharmacological inhibition of ADAM10 and ADAM17 decreased Robo4-CTF. hRobo4-HA-FLAG transiently expressing dECs were treated with vehicle control DMSO or GW (6 μM) with or without Mg132 (12 μM) or chloroquine diphosphate (CD, 50 μM). The cell lysates were probed with an anti-FLAG antibody. The hRobo4 CTF was normalized to β-actin and further normalized to the control group. GW and Mg132 decreased hRobo4-CTF. ( C ) Knockdown of ADAM10 or ADAM17 decreased Robo4 CTF. The dECs were co-transected with hRobo4-HA-FLAG and scrambled or ADAM knockdown constructs and then probed with an anti-FLAG antibody in western blot. ( D ) Inhibition of ADAM10 and ADAM17 decreased endogenous Robo4-CTF. Vehicle DMSO or GW (6 μM)-treated dECs were lysed and probed with an anti-mouse Robo4 CTF antibody in western blot. No Robo4 bands were detected in the Robo4 KO dEC cell lysate. ( E ) Robo4 co-localizes with ADAM10 and ADAM17. dECs were transiently expressed with hRobo4-HA-FLAG and stained for HA and endogenous ADAM10 or ADAM17 with corresponding antibodies. The merge yellow fluorescence shows Robo4-HA (green) co-localized with ADAM10 or ADAM17 (red). Robo4-HA-FLAG co-localized with ADAM10 or ADAM17 with overlap coefficients of 0.900 and 0.834, respectively. The data represent 3 independent experiments and are presented as mean ± SD. The student's t-test was performed for a two-group comparison. *p < 0.05; **p < 0.01.

Article Snippet: Anti-N-terminal mouse Robo4 antibody (Abcam, ab10547), anti-N-terminal human Robo4 antibody (R&D Systems, MAB2454), anti-FLAG antibody (Thermo Fisher Scientific, 14-6681-82), anti-HA antibody (Chromotek, #7c9-100), anti-intracellular Robo4 domain antibody (Santa Cruz Biotechnology, sc46497), anti-ADAM10-pro antibody (Abcam, ab39178), anti-ADAM10 antibody (Bioss, bs-3574R; LSbio, C497146-200; Novus, #NBP1-76973), anti-ADAM17 antibody (Bioss, 4236R; Novus #NBP2-61719), anti-Synecan-1 antibody (Santa Cruz Biotechnology, sc-5632), anti-actin antibody (Sigma Aldrich, A2228), anti-GAPDH antibody (R&D Systems, AF5718), polyclonal anti-Unc5B antibody (R&D Systems, AF1006), anti-N-terminal CDH5 (Thermo Fisher Scientific, 14-1441-82), anti-CD31 (BD, #550274), HRP-conjugated anti-His antibody (Alpha Diagnostic International, HISP12-HRP), HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, sc-2030), HRP-conjugated donkey anti-goat IgG antibody (Santa Cruz Biotechnology, sc-2020), and HRP-conjugated goat anti-mouse IgG antibody (Invitrogen, 62-6520).

Techniques: Inhibition, Expressing, Western Blot, Construct, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: sRobo4 generation and its role in angiogenic Slit3-Robo4 signaling. Under the unstimulated condition, the Robo4 ectodomain is constitutively cleaved by ADAM10 and ADAM17 to generate sRobo4. The generated sRobo4 blocks Slit3-Robo4 interaction, thereby inhibiting angiogenic Slit3 signaling. Meanwhile, Slit3 inhibits Robo4 shedding by inducing Robo4 internalization to shield the receptor from shedding.

Article Snippet: Anti-N-terminal mouse Robo4 antibody (Abcam, ab10547), anti-N-terminal human Robo4 antibody (R&D Systems, MAB2454), anti-FLAG antibody (Thermo Fisher Scientific, 14-6681-82), anti-HA antibody (Chromotek, #7c9-100), anti-intracellular Robo4 domain antibody (Santa Cruz Biotechnology, sc46497), anti-ADAM10-pro antibody (Abcam, ab39178), anti-ADAM10 antibody (Bioss, bs-3574R; LSbio, C497146-200; Novus, #NBP1-76973), anti-ADAM17 antibody (Bioss, 4236R; Novus #NBP2-61719), anti-Synecan-1 antibody (Santa Cruz Biotechnology, sc-5632), anti-actin antibody (Sigma Aldrich, A2228), anti-GAPDH antibody (R&D Systems, AF5718), polyclonal anti-Unc5B antibody (R&D Systems, AF1006), anti-N-terminal CDH5 (Thermo Fisher Scientific, 14-1441-82), anti-CD31 (BD, #550274), HRP-conjugated anti-His antibody (Alpha Diagnostic International, HISP12-HRP), HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, sc-2030), HRP-conjugated donkey anti-goat IgG antibody (Santa Cruz Biotechnology, sc-2020), and HRP-conjugated goat anti-mouse IgG antibody (Invitrogen, 62-6520).

Techniques: Generated

Journal: eLife

Article Title: Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators

doi: 10.7554/eLife.99172

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal anti-ADAM10 11G2 , , Diaclone: #857.800.000 , WB (1/1000).

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Marker, Sequencing, Purification, Transduction, Control, Software, Staining

Journal: Journal of Functional Morphology and Kinesiology

Article Title: Effects of Synthetic Anti-Inflammatory Sterol in CB3V-Induced Myocarditis: A Morphological Study on Heart Muscle Tissue

doi: 10.3390/jfmk1010069

Figure Lengend Snippet: Figure 5. ADAM10 immunostaining in mice CB3V-induced myocarditis. A-insert in (A) section of heart tissue of control healthy mice in which ADAM10 immunostaining was detected to a low extent; the relative image analysis by software in which a mainly high ADAM10 immunostaining was shown (red color); B-insert in (B) section of heart tissue of experimental mice treated with vehicle alone (HERF405 group) in which the ADAM10 immunostaining was shown to a greater extent with respect to both HE3286 and Dex groups; the relative image analysis by software in which a mainly low mixed to high ADAM10 immunolabeling was detected (respectively green and red color); C-insert in (C) section of heart tissue of experimental HE3286-treated mice in which ADAM10 immunostaining was evident and similar to Dex group; the relative image analysis by software in which a high mixed to a low ADAM10 immunostaining was detected (respectively red and green color); D-insert in (D) section of heart tissue of experimental Dex-treated mice in which ADAM10 immunolabeling was evident and similar to HE3286 group; the relative image analysis by software in which a mix of high and low ADAM10 immunolabeling was detected (respectively red and green color); (A–D) original magnification 40ˆ; scale bar: 50 µm; (E) A bar chart representing a comparison of the percentage areas of ADAM10-positive areas in HE3286 (n. 12), Dex (n. 12), HERF405 (n. 12)-treated mice vs. control healthy (n. 5) mice, expressed as percentage positive, dark brown pixels of the analyzed fields (total of high + low immunostaining); (F) A bar chart representing a comparison of the immunostaining intensity in ADAM10-positive areas in HE3286 (n. 12), Dex (n. 12), HERF405 (n. 12)-treated mice vs. control healthy (n. 5) mice, expressed by densitometric count (pixel2) of dark brown pixels of the analyzed fields. Red bars, high immunostaining; green bars, low immunostaining. Data are presented as mean ˘ SD. * p < 0.05; ** p < 0.01.

Article Snippet: Following blocking, the sections were incubated overnight at 4 ̋C with mouse monoclonal anti-TNFα antibody (sc-130349, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) diluted 1:100 antibody diluent (Dako, Agilent Technologies Company, Santa Clara, CA, USA); goat polyclonal Anti-IL-6 antibody (sc-1265-R, Santa Cruz Biotechnology, Inc.) diluted 1:100 in antibody diluent (Dako, Agilent Technologies Company); mouse monoclonal Anti-MMP-9 antibody (NCL-MMP9-439, Novocastra Laboratories Ltd., Newcastle upon Tyne, UK) diluted 1:100 antibody diluent (Dako, Agilent Technologies Company); rabbit polyclonal Anti-ADAM10 antibody (ab 1997, Abcam plc, Cambridge, UK) diluted 1:100 antibody diluent (Dako, Agilent Technologies Company); mouse monoclonal Anti-HSP-70 antibody (sc-24, Santa Cruz Biotechnology, Inc.) diluted 1:100 in antibody diluent (Dako, Agilent Technologies Company).

Techniques: Immunostaining, Control, Software, Immunolabeling, 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: 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