The murine lung epithelial cell line (MLE12 cells) (American Type Culture Collection, Manassas, VA, USA) was cultured in HITES medium complemented with 10% fetal bovine serum. Cells were maintained in a 37°C incubator in the presence of 5% CO2. V5 antibody was purchased from Invitrogen (Carlsbad, CA). β-actin antibody, scrambled siRNA, LPA1 siRNA, control shRNA, TrkA shRNA, batimastat (BB94), and LPA were from Sigma Aldrich (St. Louis, MO). GM6001 was from Enzo Life Science (Farmingdale, NY). Antibodies to phospho (pY674/Y675)-TrkA and Myc tag were from Cell Signaling Technology (Danvers, MA). Antibody to TrkA was from EMD MilliPore (Billerica, MA). Antibodies to phospho (p)-Erk1/2, Erk1/2, and TrkA inhibitor (TrkAi) were from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse secondary antibodies and ECL kit for detection of proteins by Western blotting was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). All other reagents were of analytical grade.
Gm6001
Manufactured by Enzo Life Sciences
Sourced in United States, France
GM6001 is a broad-spectrum matrix metalloproteinase (MMP) inhibitor. It inhibits the catalytic activity of various MMPs, including MMP-1, MMP-2, MMP-3, and MMP-9.
Automatically generated - may contain errors
Lab products found in correlation
Chloroquine, by Merck Group (2 mentions)
Batimastat bb94, by Merck Group (1 mentions)
Control shrna, by Merck Group (1 mentions)
V5 antibody, by Thermo Fisher Scientific (1 mentions)
Scrambled sirna, by Merck Group (1 mentions)
Lpa1 sirna, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit and anti mouse secondary antibodies, by Bio-Rad (1 mentions)
Mle 12 cells, by American Type Culture Collection (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Y 27632, by Merck Group (1 mentions)
Secinh3, by Bio-Techne (1 mentions)
Golgicide a, by Santa Cruz Biotechnology (1 mentions)
C3 transferase, by Cytoskeleton (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Actinonin, by Merck Group (1 mentions)
Mmp408, by Merck Group (1 mentions)
13 protocols using gm6001
1
Murine Lung Epithelial Cell Culture
2
Inhibitor Studies in Podosome Formation
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For drug inhibition studies, cells were treated with 30 µM SecinH3 (Tocris Bioscience), 10 µM Golgicide A (Santa Cruz Biotechnology, Inc.), 5 µg/ml BFA (Sigma-Aldrich), 30 µM Y-27632 (Sigma-Aldrich), 25 µM GM6001 (Enzo Life Sciences), and 2 µg/ml C3 transferase (Cytoskeleton, Inc.) in complete medium for 1–2 h or 4 h for GM6001 at 37°C with 5% CO2 and subsequently fixed with 4% PFA. For live-cell imaging, cells were imaged immediately after addition of appropriate inhibitors, which remained in the medium during the entire period of image acquisition. To study the effect of inhibitors on podosomes formed by MEFs plated on RGD lipid bilayer, the cells were treated with appropriate inhibitors 30–45 min after cell seeding on the bilayer.
3
Glioma Cell Culture and Hypoxia Conditions
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Human D54MG, U373MG and SNB191 glioma cell lines and primary human GBM XD45 and JX10 xenolines (UAB Brain Tumor Specialized Program Of Research Excellence) were cultured in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Paisley, UK) supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine, penicillin/streptomycin and non-essential amino acids (all Gibco-BRL) (27 (link),28 (link)). The cells were cultured at 37°C, in a humidified atmosphere of 5% CO2 and 95% air (~21% pO2). For the hypoxia experiments, the cells were kept for the indicated durations in a cell culture incubator (I-Glove; BioSpherix, Inc., Lacona, NY, USA) with an oxygen level set to 1 or 5% pO2, as indicated. Chloroquine was purchased from Sigma-Aldrich (St. Louis, MO, USA) and the wide-spectrum MMP-inhibitor, GM6001, was obtained from Enzo Life Sciences Inc., (Farmingdale, NY, USA).
4
Recombinant Protein Purification Protocols
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Recombinant His‐tagged sizzled (from Xenopus laevis) was produced in 293‐EBNA cells and purified as described previously 7 (purity > 95% (SDS/PAGE), ESI‐MS m/z calcd 35481.8 found 35483.2). S33A was synthesized as described in 13 (purity > 97% (HPLC); HRMS (high‐resolution mass spectrometry; ESI (electrospray ionisation)) m/z: [MH+] calcd for C27H33N4O6S 541.2120; found 541.2119). FG‐2575 was designed and prepared by FibroGen, Inc. (purity > 95% (HPLC); MW = 503.6251; HRMS (ESI) m/z: [MH+] within 1.6 ppm of calcd value). The synthesis of the phosphinic peptide RXP‐1001 will be described elsewhere (Lecorché et al., in preparation; purity > 95% (HPLC); MS (matrix‐assisted laser desorption ionization) m/z: [MH+] calcd for C55H67N8O14P 1095.46; found 1095.57). UK383,367 and actinonin were purchased from Sigma‐Aldrich (L'isle d'abeau, France) and GM6001 from Enzo Life Sciences (Villeurbanne, France). All commercial compounds have purity > 98% (HPLC or TLC). Stock solutions of the inhibitors were prepared at 10 mm in DMSO, except for RXP‐1001 (3 mm in EtOH 50%) and sizzled (120 μm in HEPES 20 mm NaCl 0.5M pH 7.4).
5
Analytical Characterization of Small Molecules
6
Cytoskeletal Protein Antibody Validation
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Cortactin (sc-30771), Tks5 (sc-30122), and Arp2 (sc-H-84) antibodies were from Santa Cruz Biotechnology. Cortactin (ab33333) was from Abcam. Anti-actin (Millipore), anti-GFP (Rockland), Rac1/3 (Upstate), specific anti-Rac3 (ProteinTech Group), and anti-Rac1 (Upstate) were also used. All Alexa Fluor–conjugated secondary antibodies were obtained from Molecular Probes (Life Technologies). GM 6001 was from Enzo Life Sciences.
7
Cultured Rat Hippocampal Neuron Activation
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Cultured hippocampal neurons were prepared from embryonic day 18 Sprague-Dawley rat brains. Dissociated neurons on poly-l -lysine coated (1 mg ml−1) coverslips were placed in neurobasal medium supplemented with B27 (Invitrogen), 0.5 mM l -glutamate and penicillin–streptomycin. For activation, cultured neurons at days in vitro (DIV) 18–21 were treated with NMDA (Sigma; 20 µM, 3 min), KCl (Sigma; 50 mM, 5 min) or l -glutamate (Sigma; 50 µM, 30 min). For induction of NMDA receptor-dependent LTD or mGluR-LTD, cultured neurons were treated with NMDA (20 µM, 3 min) and DHPG (Tocris; 50 μM, 30 min), respectively, and returned to normal conditioning medium. For the blockade of NMDA receptors, cultured neurons were pretreated with APV (Tocris; d -2-amino-5-phosphovalerate; 50 µM, 30 min), followed by stimulation with NMDA (20 µM, 3 min) in the presence of APV or with l -glutamate (50 µM, 1 min) or KCl (30 mM, 1 min) in the absence of APV. For chronic neuronal inhibition or activation, neurons were treated with tetrodotoxin (Tocris; TTX; 1 µM, 48 h) or with bicuculline (Tocris; Bic; 10 µM, 36 h). For blockade of metalloproteinase and γ-secretase activity, neurons were pretreated with GM6001 (Enzo Life Sciences; 2.5 and 25 µM, 30 min), DAPT (Sigma; 250 nM, 2 h; 2 µM, 3 h) and L-685,458 (Calbiochem; 1 µM, 30 min) before and during NMDA stimulation (20 µM, 3 min).
8
Tamoxifen and Estrogen Receptor Signaling
9
Extracting and Quantifying Knee Collagen Biomarkers
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Protein was extracted from knee joints according to Nielsen et al (24 (link)) at week 0, 1, 2 and 4 after operation. Knee joints were isolated after euthanasia as described above, then immediately frozen in liquid nitrogen and stored at −80°C for use. The tibia and femur were cut 3 mm from the joint, producing samples weighing 500–700 mg. These samples were frozen in liquid nitrogen again, and transferred to a Bessman tissue pulverizer (Spectrum Chemical Manufacturing Corp.). The samples were crushed with 3 ml extraction buffer, consisting of 50 mM Tris-HCl buffer (pH 7.4), 0.1% Triton X-100, 0.1 M NaCl, 10 Mm (GM6001; Enzo Life Sciences, Inc.) and 1 tablet/10 ml buffer of Complete Mini EDTA-free protease inhibitor cocktail (Roche Diagnostics). Tissues were homogenized twice for 30 sec using an OMNI homogenizer (Omni International, Inc.) at speed level 4, then the samples were centrifuged for 10 min at 1,700 × g and 4°C, supernatants were obtained and centrifuged at 10,000 × g and 4°C. Finally, the supernatants were stored at −20°C until use.
Rat-specific commercially available ELISA kits (Elabscience®) were used to evaluate the levels of C-terminal telopeptide of type I collagen (CTX)-I (cat no. E-EL-R1456) and CTX–II (cat no. E-EL-R2554) in protein extracts collected from the knee, according to the manufacturer's instructions (24 (link)).
Rat-specific commercially available ELISA kits (Elabscience®) were used to evaluate the levels of C-terminal telopeptide of type I collagen (CTX)-I (cat no. E-EL-R1456) and CTX–II (cat no. E-EL-R2554) in protein extracts collected from the knee, according to the manufacturer's instructions (24 (link)).
10
TREM2 Antibody Treatment on hiMGL Cells
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Eight hours after seeding the cells, they were treated with anti‐human TREM2 antibodies (Fig EV1C ). Antibodies were diluted in RPMI medium and added to the cells with a final concentration of 20 µg/ml. As control for TREM2 shedding, cells were treated with GM6001 (25 µM, Enzo Life Sciences), or DMSO as a vehicle control. hiMGL were seeded in six‐well plates with 400,000 cells/well. Eight hours after seeding, antibodies were diluted in iMGL media and added at a concentration of either 20 or 40 µg/ml. Isotype or TREM2 antibodies were added at a concentration of 40 µg/ml and 24 h after antibody treatment, medium and cells were harvested as previously described.
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