LOX enzymes were purchased from OriGene (Rockville, MD, USA). Matrix metalloproteinase-2 (MMP-2) and cathepsin B enzymes were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). The LOX inhibitor, β-aminopropionitrile, was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Invitrogen Canada (Burlington, ON, Canada), and RPMI 1640 medium was obtained from Gibco BRL (Gaithersburg, MD, USA). For Western blot analysis, anti-LOX and a horseradish peroxidase-conjugated anti-mouse antibody were purchased from Aviva Systems Biology (San Diego, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. A commercially available LOX assay kit, AmpliteTM fluorimetric LOX Assay kit, was purchased from AAT Bioquest (Sunnyvale, CA, USA). HAuCl4 and tri-sodium citrate were purchased from Sigma-Aldrich and Fisher Scientific (Waltham, MA, USA), respectively.
β aminopropionitrile
Manufactured by Merck Group
Sourced in United States
β-aminopropionitrile is a chemical compound that serves as a laboratory reagent. It has the molecular formula C3H6N2. This product is typically used in various chemical and biochemical research applications, though its specific functions and intended uses should not be extrapolated upon.
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Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Haucl4, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Trisodium citrate, by Merck Group (1 mentions)
Batimastat, by Abcam (1 mentions)
S 4 nitro blebbistatin, by Cayman Chemical (1 mentions)
4 pba, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
β apn, by Merck Group (1 mentions)
Zoletil 50, by Virbac (1 mentions)
Val5 ang 2, by Merck Group (1 mentions)
C57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
Alzet micro osmotic pump, by Durect (1 mentions)
Rat tail collagen, by Thermo Fisher Scientific (1 mentions)
Pd325901, by MedChemExpress (1 mentions)
P fak y397, by Abcam (1 mentions)
Pkh26 red fluorescent cell linker mini kit, by Merck Group (1 mentions)
9 protocols using β aminopropionitrile
1
Characterization of Enzymatic Interactions in Cancer
2
Inhibition of Cellular Contractility
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Batimastat was purchased from abcam (ab142087), (S)‐4′‐nitro‐Blebbistatin from Cayman Chemical (24171), β‐aminopropionitrile from Sigma Aldrich (A3134).
3
Inducing Thoracic Aortic Aneurysm in Mice
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Wild-type or CHOP knockout mice of C57BL/6 background were obtained from the Jackson Laboratory (Bar Harbor, ME) and fed in a specific pathogen free (SPF) animal facility in Beijing Anzhen Hospital. The TAAD mice model was induced as described recently [3 (link)]. In brief, 3-week-old male mice were given a normal diet and administered a solution of BAPN (β-aminopropionitrile, Sigma–Aldrich, St. Louis, MO), which was dissolved in drinking water at a concentration of 1 g/kg per day for 4 weeks. BAPN treatment led to TAAD formation via inhibiting lysyl oxidase activity, which is responsible for collagen and elastin cross-linking. To assess the effect of an ER stress inhibitor, 4-phenylbutyric acid (4-PBA, Sigma) was administered via intraperitoneal injection at a concentration of 200 mg/kg per day. All studies were approved by the Institutional Animal Care and Use Committee of Beijing Anzhen Hospital, Capital Medical University, Beijing, China. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.
4
Collagen Synthesis Estimation in Breast Stromal Fibroblasts
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A modification of the protease-free collagenase method was employed for collagen synthesis estimation, as described previously [79 (link)]. Briefly, breast stromal fibroblasts at approximately 90% confluency were incubated for 24 h in DMEM containing 0.1% (v/v) FBS. Then fresh medium containing 5 μCi/ml L-[3H]proline (31 Ci/mmol; Moravek Biochemicals Inc., CA, USA), 50 μg/ml β-aminopropionitrile (Sigma), 50 μg/ml ascorbic acid (Sigma), and 0.1% (v/v) FBS was added for another 48 h. The proteins secreted in the conditioned medium were precipitated with 10% (v/v) trichloroacetic acid (TCA) and re-dissolved in 0.2 N NaOH. One half of each sample was digested with 5 bovine tendon collagen units/ml protease-free collagenase from Clostridium histoliticum (EC number 3.4.24.3; Sigma), while the other half was left untreated, to be used as blank. The undigested protein was precipitated with TCA in the presence of tannic acid, and the radioactivity of the supernatant was measured using a β-counter. Collagen synthesis rate for each sample was calculated after subtraction of the blank value and normalization according to the cell number.
5
Avoidance of Traumatic Tooth Extraction
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For the surgical intervention, the animals were anesthetized by administering Zoletil 50 (Virbac Lab, Carros, France) intramuscularly at a dose of 100–150 mg/kg and underwent a 5-day treatment with 0.4% β–aminopropionitrile (β-APN; Sigma-Aldrich, St. Louis, MO, USA) to avoid traumatic extraction [15 (link)]. Forty bilateral maxillary first molars with complete root formation were extracted from 20 animals. Preoperatively, the oral cavity of the animals was cleaned with 2% chlorhexidine solution. Extraction of the maxillary first molars was performed gently using sterile extraction forceps. After excluding any roots that fractured during extraction, the allotment of 10 teeth per group was maintained by including additional experimental animals. The extracted teeth were exposed to a dry environment for 5 minutes at room temperature or preserved in HBSS solution for 60 minutes; subsequently, the teeth and sockets were cleaned with saline prior to replantation. After replantation, a single dose of 20,000 IU of penicillin G (Alvogen potassium penicillin G, Alvogen, Seoul, Korea) was administered intramuscularly in all animals, and they were fed a soft diet for 7 days [7 (link)]; euthanasia was performed 8 weeks after replantation (Figure 1 ).
6
Angiotensin II and Vascular Stiffness in Mice
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Three-month-old male C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). This study was approved by the University of Arizona Animal Care Committee and conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). All mice were randomly divided into placebo and three treatment groups.
A two by two study design was used to determine the effects of Ang II and BAPN on vascular stiffness. The BAPN (β-aminopropionitrile, Sigma-Aldrich) groups were pre-treated for 1 week prior to any test conducted. BAPN was dissolved into drinking water at a dosage of 1.5g/L ad libitum. Ang II was administered with a subcutaneous Alzet micro-osmotic pump (Durect Corporation, Cupertino, CA) releasing the Ang II receptor type I (AT1) agonist [Val5]Ang II (Sigma) at a rate of 490ng/kg/min. Control groups also received the pumps filled with vehicle (PBS). On Day 14, the mice were sacrificed for descriptive analyses.
A two by two study design was used to determine the effects of Ang II and BAPN on vascular stiffness. The BAPN (β-aminopropionitrile, Sigma-Aldrich) groups were pre-treated for 1 week prior to any test conducted. BAPN was dissolved into drinking water at a dosage of 1.5g/L ad libitum. Ang II was administered with a subcutaneous Alzet micro-osmotic pump (Durect Corporation, Cupertino, CA) releasing the Ang II receptor type I (AT1) agonist [Val5]Ang II (Sigma) at a rate of 490ng/kg/min. Control groups also received the pumps filled with vehicle (PBS). On Day 14, the mice were sacrificed for descriptive analyses.
7
Extracellular Matrix Remodeling Assay
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The following antibodies were used: Col1a2 (14695), FN1 (15613) and GAPDH (10494) from ProteinTech (USA); MMP-9 (AF909, R&D); MMP-10 (NB100–92182, Novus); p-FAK (Y397, ab39967), LOX (ab174316) and Paxillin (ab32084) from Abcam; p-Erk1/2 (Thr202/Tyr204, 4377), p-Src (Y416, 2101), AlexaFluor goat anti-rabbit IgG (594 conjugate, 8889) and sheep anti-rabbit HRP-linked IgG (7074) from Cell Signaling. Reagents included β-aminopropionitrile (BAPN), PKH26 Red Fluorescent Cell Linker Mini Kit (Sigma), PD-325901 (MedChem Express), Hydroxyproline Detection Kit (Nanjing Jiancheng Bioengineering Institute), Rat tail collagen (Invitrogen), Cell counting Kit-8 (Dojindo) and Matrigel matrix (BD).
8
Modulation of Fibroblast Responses
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rhIL-17A, rhTGF-β, monoclonal mouse IgG1 TGF-β1, 2, 3 antibody, IL-6, MCP-1, MMP-1, IL-8, pro-collagen Iα1 and fibronectin ELISA DuoSet kits were from R&D Systems (Abingdon, UK); DMEM, PBS, glutamine, penicillin, streptomycin, trypsin, dispase, collagenase type I from Gibco (Paisley, UK); FCS from Biowest (Nuaillé, France); BSA, α-ketoglutaric acid, β-amino propionitrile, l -ascorbic acid, p38 MAPK inhibitor SB203580, and PI3K inhibitor LY294002 from Sigma (St. Louis, MO, USA); MEK1/2 inhibitor U-0126 from Calbiochem (San Diego, CA, USA); TGF-βRI inhibitor SD 208, JNK inhibitor SP 600125 and IKK-2 inhibitor TPCA-1 from Tocris Bioscience (Bristol, UK); LEAF irrelevant control mAbs from Biolegend (San Diego, CA, USA); Complete Protease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor from Roche (Basel, Switzerland); nitrocellulose membranes and chemiluminescence (ECL) blotting analysis system from GE Healthcare (Zurich, Switzerland); phospho-Akt (Ser473), phospho-Smad2 (Ser465/467), phospho-p38 MAPK (Thr180/Tyr182), phospho-NF-κB p65 (Ser536), phospho-IκB-α (Ser32), β-actin and BSA for Western blots from Cell Signaling (Danvers, MA, USA); TMB ELISA substrate from Abcam (Cambridge, UK); EZ4U cell proliferation assay from Biomedica (Vienna, Austria).
9
Immunohistochemistry for IL-17 Cytokines
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Polyclonal goat anti-human IL-17A Ab, mouse anti-human IL-17C, IL-17E and IL-17F mAbs, recombinant human tumor necrosis factor (TNF), transforming-growth factor (TGF), IL-17A, IL-17C, IL-17E and IL-17F were from R&D Systems (Abingdon, UK); mouse anti-human mast cell tryptase mAb (clone AA1), polyclonal rabbit anti-mouse and goat anti-mouse immunoglobulins biotinylated from Dako (Glostrup, DK); monoclonal mouse anti-human CD3 (clone PS1) from Novocastra (Newcastle, UK); Alexa-488-conjugated donkey anti-goat and alexa-568 conjugated donkey anti-mouse or anti-rabbit, Dulbecco's modified Eagle's medium (DMEM), phosphate buffered saline (PBS) glutamine, penicillin, streptomycin, trypsin, sodium pyruvate and fetal calf serum were from Life technologies (Paisley, UK). Tyramide, 3,3-Diaminobenzidine (DAB), α-ketoglutaric acid, β-amino propionitrile and L-ascorbic acidwere from Sigma (St. Louis, MO); Vectastain elite ABC kit and Vectashield with DAPI from Vectorlab (Peterborough, UK). All the antibodies have been used at the final concentration of 2.5 µg/ml.
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