4 methylumbelliferone 4 mu

Manufactured by Merck Group

Sourced in United States

4-methylumbelliferone (4-MU) is a fluorescent compound used as a label or indicator in various laboratory techniques. It exhibits blue-green fluorescence when excited by ultraviolet light. 4-MU is commonly used as a substrate for enzymatic assays, particularly those involving glycosidases and phosphatases.

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Lab products found in correlation

Alamethicin, by Merck Group (3 mentions) 850 fluorescence spectrophotometer, by Hitachi (3 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions) Bxpc 3, by American Type Culture Collection (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Panc 1, by American Type Culture Collection (2 mentions) Aspc 1, by American Type Culture Collection (2 mentions) Capan 2, by American Type Culture Collection (2 mentions) Cfpac 1, by American Type Culture Collection (2 mentions) Nor p1, by RIKEN BioResource Center (2 mentions) Propofol, by Merck Group (2 mentions) Zidovudine, by Merck Group (2 mentions) 4 methylum belliferyl β d glucuronide hydrate 4 mu g, by Merck Group (2 mentions) Formic acid, by Thermo Fisher Scientific (2 mentions) Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions) Spectramax id5 plate reader, by Molecular Devices (1 mentions) Protein assay kit, by Bio-Rad (1 mentions) Bovine serum albumin (bsa), by Bio-Rad (1 mentions) Mia paca 2, by American Type Culture Collection (1 mentions) Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)

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4-methylumbelliferone (52 citations) Methylumbelliferone (4 citations)

35 protocols using 4 methylumbelliferone 4 mu

Quantifying PPT1 Inhibitor Activity in HepG2 Cells

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HepG2 cells were
obtained from ATCC and were cultured in Dulbecco’s modified
Eagle medium + 10% FBS at 37 °C calcium and 5% CO₂ level. To test PPT1 inhibition in cells, HepG2 cells were seeded
in 6-well plates and left overnight to adhere. The following day,
compounds were added to cell culture media and incubated for 5 h,
followed by cell lysis and protein extraction. The MPER extraction
agent (Thermo Fisher Scientific) along with the Halt protease inhibitor
(Thermo Fisher Scientific) in 0.5% Triton X100 were used for cell
lysis followed by centrifugation at 14,000 rpm for 15 min to obtain
cell lysates. Lysates were normalized for protein amount and 5 μL
of cell lysates were used for the PPT1 assay as described earlier
with modifications.^{42 (link),47 (link)} 4-methylumbelliferone (4-MU)
(Sigma-Aldrich) was used to make a standard curve. Fluorescence intensity
was measured using a SpectraMax iD5 plate reader (Molecular Devices).
For testing PPT1 inhibitor activity in cell lysates, 50 μL of
HepG2 cell lysates were treated with the compounds for an hour, followed
by enzyme inhibition using the PPT1 enzyme assay as above. Experiments
were performed in biological duplicates containing technical replicates
in triplicates. The percent PPT1 activity was determined relative
to that of the DMSO control.

Puhl A.C., Raman R., Havener T.M., Minerali E., Hickey A.J, & Ekins S. (2024). Identification of New Modulators and Inhibitors of Palmitoyl-Protein Thioesterase 1 for CLN1 Batten Disease and Cancer. ACS Omega, 9(10), 11870-11882.

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Renal Biomarker Measurement Protocol

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Serum creatinine level was determined using synchron creatinine reagent (Bechman Coulter, Carlsbad, CA, USA). Blood urea nitrogen (BUN) level was determined using the Beckman BUN kit (Sigma, St. Louis, MO, USA). Urinary N-acetyl-β-D-glucosaminidase (NAG) was measured using the fluorescent 4-methylumbelliferone (4-MU) (Sigma, St. Louis, MO, USA) [29 (link),30 (link)]. The substrate (4-methylumbelliferyl N-acetyl-β-d-glucosaminide) was split via NAG to form 4-MU and 4-MU, reflecting the NAG activity, and the absorbance was measured at 370/460 nm using a plate reader (BMG Labtech Inc., Offenburg, Germany).
Urinary protein level was determined using Bio-Rad protein assay kits (Bio-Rad, Hercules, CA, USA) according to the Bradford method [31 (link)]. Bovine serum albumin (Bio-Rad Inc., Herecules, CA, USA) was used as a standard solution, and the protein content was positively correlated with the O.D. at 590 nm.

Chou C.K., Huang Y.S., Lin P.Y., Imai K., Chen S.M, & Lee J.A. (2021). The Ginsenoside Rg1 Rescues Mitochondrial Disorders in Aristolochic Acid-Induced Nephropathic Mice. Life, 11(10), 1018.

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Pancreatic Ductal Adenocarcinoma Cell Lines

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We used 10 PDAC cell lines, AsPC-1, BxPC-3, Capan-2, CFPAC-1, MIAPaCa-2, PANC-1, SW1990 (American Type Culture Collection, Manassas, VA, USA), KP-2, KP-3 (JCRB Cell Bank, Osaka, Japan), and NORP-1 (RIKEN BRC Cell Bank, Tsukuba, Ibaraki, Japan). An immortalized cell line derived from human pancreatic duct, HPDE, was a kind gift from Dr. M.S. Tsao (Dept. of Pathology, Univ. of Toronto, Canada). PDAC cell lines were maintained in RPMI1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and 1% streptomycin and penicillin (Life Technologies). HPDE was maintained in HuMedia-KG2 (KURABO, Osaka, Japan), in a 5% CO² incubator at 37° C. 4-Methylumbelliferone (4-MU) and dextran sulfate were purchased from SIGMA-ALDRICH Corp. (St. Louis, MO, USA).

Kudo Y., Kohi S., Hirata K., Goggins M, & Sato N. (2019). Hyaluronan activated-metabolism phenotype (HAMP) in pancreatic ductal adenocarcinoma. Oncotarget, 10(54), 5592-5604.

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Hyaluronan Synthesis Inhibition in Ovarian Cancer Cells

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ES-2 and ES-2:ES-2-Rv-NICD3 cells were plated at 2 × 10⁵ cells/well in 6 well plates and cultured 48 h before treatment with the hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU, 1 mM, Sigma-Aldrich) or vehicle control (PBS) for 24 h [37 (link)]. Protein lysates from monolayer and spheroids were prepared in RIPA buffer as described previously [59 (link)]. ES-2, A2780 and OVCAR3 cells were cultured to 80% confluency before protein isolation. 20 µg of protein was electrophoresed on 4–20% TGX gels (Bio-Rad, Hercules, CA, USA) and transferred overnight to PVDF membrane (GE healthcare, little Chalfont, England). Proteins were detected with DAB2 rabbit monoclonal antibody (1/2000, ab256524, Abcam), anti-rabbit IgG peroxidase conjugated antibody (1/4000, Sigma-Aldrich) and Amersham™ ECL™ Prime (Cytvia, Marlborough, MA, USA). Chemiluminescence was detected using the ChemiDoc™ Imaging System (Bio-Rad) and band intensity was calculated with ImageLab software (Bio-rad, version 6.1). β-actin monoclonal mouse antibody (1/5000, ab8226, Abcam) or GAPDH monoclonal mouse antibody (1/50,000, 60004-1-Ig, Proteintech®, Rosemont, IL, USA) was used as a loading control.

Price Z.K., Lokman N.A., Sugiyama M., Koya Y., Yoshihara M., Oehler M.K., Kajiyama H, & Ricciardelli C. (2023). Disabled-2: a protein up-regulated by high molecular weight hyaluronan has both tumor promoting and tumor suppressor roles in ovarian cancer. Cellular and Molecular Life Sciences, 80(11), 320.

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Cell Culture and Cytokine Treatment

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The MCF-7 breast cancer cell line and the A549 lung adenocarcinoma cell line were obtained from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (HyClone, Logan, UT, USA) and 100 U/ml of penicillin and 100 mg/l of streptomycin at 37°C in a 5% CO₂ humidified atmosphere. Logarithmic phase cells were used in the experiments. In some cases, 10% fetal calf serum was changed to serum-free medium depending on the experiment. The cells were treated with EGF at 10 ng/ml or TGF-β1 at 5 ng/ml for 24 h. EGF (AF-100-15) and TGF-β1 (AF-100-18B) were purchased from PeproTech (Rocky Hill, NJ, USA). 4-Methylumbelliferone (4-MU) was obtained from Sigma-Aldrich (St. Louis, MO, USA).

LI L., QI L., LIANG Z., SONG W., LIU Y., WANG Y., SUN B., ZHANG B, & CAO W. (2015). Transforming growth factor-β1 induces EMT by the transactivation of epidermal growth factor signaling through HA/CD44 in lung and breast cancer cells. International Journal of Molecular Medicine, 36(1), 113-122.

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Effects of HA and 4-MU on MG Cell Proliferation

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The effect of HA on MG cell proliferation was assayed in vitro by measuring the incorporation of 5-bromo-2-deoxyuridine (BrdU) into the newly synthesized DNA of replicating cells (BrdU Cell Proliferation Kit, EMD Millipore, Darmstadt, Germany). The hMGCs were plated at a seeding density of 5000 cells/well in 96-well plates and allowed to grow for 24 hours in the presence or absence of HA or 4-methylumbelliferone (4-MU; Sigma-Aldrich Corp.). The concentrations used were 0.02% and 0.05% of HMWHA; 0.02% and 0.05% of LMWHA; or 0.25 mM, 0.5 mM, or 2 mM of 4-MU. After 24 hours, with the cells in their exponential growth phase, BrdU was added to each well and left for 2 hours. The cells were then fixed and the detection of BrdU incorporation carried out according to the manufacturers' instructions. Absorbance in each well was read as optical density (OD) by using a spectrophotometer microplate reader (Fluostar Omega, BMG Labtech, Offenburg, Germany) set at a test wavelength of 450 nm. Each condition was tested in triplicate and three separate experiments were carried out. The mean of each individual experiment is presented as a single point on the graph.

Sun M., Puri S., Parfitt G.J., Mutoji N, & Coulson-Thomas V.J. (2018). Hyaluronan Regulates Eyelid and Meibomian Gland Morphogenesis. Investigative Ophthalmology & Visual Science, 59(8), 3713-3727.

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Synthesis and Glucuronidation of NAF Enantiomers

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R(+)-NAF (>99.5% ee) and S(-)-NAF (>99.5% ee) (Figure 1) were synthesized according to previous methods (Shivani et al., 2007 (link)). The internal standard (IS) Z10 (CN201110148322.7) was obtained from our laboratory. HPLC-grade methanol, acetonitrile, and acetic acid were obtained from Merck (Darmstadt, Germany). Uridine diphosphate glucuronic acid (UDPGA), alamethicin, Tris, magnesium chloride, methylum-belliferone (MU), 4-methylumbelliferone (4-MU), 4-methylum-belliferyl-β-D-glucuronide hydrate (4-MU-G), propofol, propofol glucuronide, zidovudine, zidovudine glucuronide, fluconazole, and niflumic acid, trifluoperazine dihydrochloride (TFP), and TFP-glucuronide (TFP-G) were purchased from Sigma–Aldrich (St. Louis, MO, United States). Recombinant human UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) and the commercially available microsomes from human liver (HLMs), intestine (HIMs), and kidney (HKMs), as well as those from rat liver (RLMs), intestine (RIMs), and kidney (RKMs), were purchased from BD Gentest (Woburn, MA, United States). Protein contents of the microsomes were used according to the manufacturers’ instructions. Ultrapure water was used in all experiments (Millipore, Billerica, MA, United States).

Liu X.W., Rong Y., Zhang X.F., Huang J.J., Cai Y., Huang B.Y., Zhu L., Wu B., Hou N, & Luo C.F. (2018). Human UDP-Glucuronosyltransferase 2B4 and 2B7 Are Responsible for Naftopidil Glucuronidation in Vitro. Frontiers in Pharmacology, 8, 984.

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TGF-β2, 4-MU, and HAPLN1 Protocol

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Recombinant human TGF-β2 was purchased from R&D Systems (302-B2-010/CF, Minneapolis, MN, USA). 4-Methylumbelliferone (4-MU) and hyaluronic acid (HA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Recombinant human HAPLN1 was purchased from Cusabio (Houston, TX, USA).

Ha H.C., Zhou D., Fu Z., Back M.J., Jang J.M., Shin I.C, & Kim D.K. (2023). Novel Effect of Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1) on Hair Follicle Cells Proliferation and Hair Growth. Biomolecules & Therapeutics, 31(5), 550-558.

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Fluorogenic Assay for rIDUA_RLT Activity

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Turnip hairy root culture media coming from transformed hairy root cultures as well as the purified rIDUA_RLT protein were used to determine the activity of the recombinant protein of interest by using the fluorogenic substrate sodium 4‐methylumbelliferyl‐α‐L‐Iduronide (4MU‐I; Santa Cruz Biotechnology) as described in (Ou et al., 2014). The 4MUI substrate was diluted to a working solution of 400 μm 4MU‐I with the reaction buffer 0.4 m sodium formate, pH 3.5. Twenty‐five μL of sample were added to 25 μL of 400 μm 4MU‐I substrate. The mixture was incubated at 37 °C for 30 min and 200 μL glycine carbonate buffer (pH 9.8) was added to quench the reaction. 4‐Methylumbelliferone (4MU) (Sigma) was used to prepare the standard calibration curve. Fluorescence was measured using a plate reader (TECAN Infinite M1000, Männedorf, Switzerland) with excitation at 355 nm and emission at 460 nm. IDUA enzyme activity was expressed in units (μmol converted to product per minute) per sample volume (millilitres). The parameters K_M, k_cat and V_max were calculated by linear fit on a Lineweaver‐Burk plot (Ou et al., 2014).

Cardon F., Pallisse R., Bardor M., Caron A., Vanier J., Ele Ekouna J.P., Lerouge P., Boitel‐Conti M, & Guillet M. (2018). Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha‐L‐iduronidase. Plant Biotechnology Journal, 17(2), 505-516.

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Quantifying β-Galactosidase Activity in Plant Extracts

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β-gal activity in plant crude extracts was determined using the fluorogenic substrate 4-methylumbelliferyl β-D-galactopyranoside (4MUGal, Sigma). Frozen infiltrated N. benthamiana leaves were ground in 1:2 w/v of tissue to buffer (100 mM Tris, 150 mM MgCl₂, 10 mM Na₂S₂0₅, pH 7.5 containing 0.1% Tween 20) and centrifuged at 16,000xg for 30 min at 4°C. This crude extract was diluted 1:10 in enzyme buffer (100 mM sodium acetate buffer, pH 4.3). Ten μl of the diluted sample was loaded onto a MicroFluor2 black 96-well plate (Thermo-Fisher) and then mixed with 20 μl/well of substrate solution (1 mM 4MUGal in 100 mM sodium acetate buffer, pH 4.3). The same procedure was done with crude extracts from plants infiltrated with an “empty vector” (pBIB-Kan) to serve as a blank. After addition of the substrate, the plate was wrapped in aluminum foil and incubated at 37°C for 30 min. The reactions were terminated by the addition of 270 μl of stop solution (0.2 M glycine, 0.125 M sodium carbonate, pH 10.7). Serial dilutions of 4-methylumbelliferone (4MU) (Sigma) from 0.07 to 4.5 nM were used in order to establish a standard curve. Fluorescence was measured on the PolarStar (BMG Labtech) using 350 and 420 nm for excitation and emission, respectively.

Condori J., Acosta W., Ayala J., Katta V., Flory A., Martin R., Radin J., Cramer C.L, & Radin D.N. (2015). Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel β-galactosidase:RTB lectin fusion. Molecular genetics and metabolism, 117(2), 199-209.

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