C. albicans cells were grown in yeast extract/peptone/dextrose broth (YPD) made according to standard recipes [51 ]. Cells were inoculated from single colonies growing on YPD plates into 20 ml YPD and grown under shaking at 30°C until the culture attained an OD600 value of 2.00 A. Once the culture had reached OD600≈2.0 A, cells were harvested and then resuspended in fresh media at a concentration of 3×107 cells/ml (OD600≈1.26 A). For blastospore induction, cells from the original culture were resuspended in fresh YPD, transferred to a sterile flask, and then grown under shaking at 30°C for 3 hours. For hyphal induction, harvested cells were resuspended in either YPD + 10% fetal bovine serum (HyClone) pre-warmed to 37°C (YPD + FBS) or YPD + N-acetylglucosamine at a concentration of 0.5 g/l GlcNAc (Sigma-Aldrich;YPD+GlcNAc), transferred into a fresh flask, and placed in an incubator with shaking at 37°C for 3 hours [11 (link), 12 (link), 42 (link), 43 (link), 52 (link)].
Glcnac
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
GlcNAc is a carbohydrate molecule that serves as a building block for various biomolecules. It is a fundamental component of the polymer chitin, which is found in the cell walls of fungi and the exoskeletons of arthropods. GlcNAc is also an important monosaccharide in the glycosylation of proteins and lipids, playing a role in various biological processes.
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Lab products found in correlation
Galnac, by Merck Group (6 mentions)
Pugnac, by Merck Group (4 mentions)
Chitin, by Merck Group (3 mentions)
Galactose, by Merck Group (2 mentions)
Streptomyces plicatus chitinase, by Merck Group (2 mentions)
Mannac, by Merck Group (2 mentions)
Glucose, by Merck Group (2 mentions)
Lsrii analyzer, by BD (1 mentions)
Mini opticon real time pcr machine, by Bio-Rad (1 mentions)
Sypro orange, by Merck Group (1 mentions)
El800, by Agilent Technologies (1 mentions)
Sheep blood, by Thermo Fisher Scientific (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
N acetyl d glucosamine, by Merck Group (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Biotin, by Merck Group (1 mentions)
Glycol chitosan, by Merck Group (1 mentions)
Chit600, by Thermo Fisher Scientific (1 mentions)
Chitosan chit100, by Thermo Fisher Scientific (1 mentions)
47 protocols using glcnac
1
Candida albicans Morphological Transition Induction
2
GlcNAc Uptake and Efflux in Rice Seedlings
3
Enrichment of O-GlcNAc Proteins from ARPE-19 Cells
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pSBTet-AAK1-GFP WT ARPE-19 cells or WT ARPE-19 cells were seeded onto 10 cm dishes until 80 to 85% confluent. Then, cells were homogenized in 400 μl cold lysis buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 2 mM PMSF, 0.5% Nonidet P-40, 25 mM TMG). After a centrifugation step (10 min at 14,000 rpm at 4 °C), the supernatant was incubated in 150 μl of WGA Agarose from Vector Biolabs (catalog #: VECTAL10235) and INPUT samples were collected. The agarose-bound WGA was prewashed with PBS containing 0.2% Nonidet P-40 (NP-40). The WGA and protein lysate were incubated overnight at 4 °C under rotation. Then, supernatant fractions were collected, followed by washing of agarose beads with 0.2% NP-40 in PBS. The O-GlcNAc–modified proteins were eluted from the WGA agarose using 1 M GlcNAc (Sigma-Aldrich) in 0.2% NP-40 in PBS at room temperature in Figure 4 . After this, the samples were resolved by Western blotting, as described above.
4
Bladder Cancer Cell Lines Protein Stability
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Bladder cancer cell lines including HT-1376, 5637, and T24, and normal uroepithelium cell line SV-HUC-1 were all purchased from ATCC (VA, USA). HT-1376 and were grown in Eagle’s Minimum Essential Medium (No. 30–2003, ATCC, VA, USA). Five thousand six hundred and thirty-seven cells were cultured in RPMI-1640 medium (Biological Industries, Kibbutz BeitHaemek, Israel), T24 cells were cultured in McCoy’s 5a Medium (No. 30–2007, ATCC, VA, USA). The base medium for SV-HUC-1 cell line is ATCC-formulated F-12K Medium (No. 30–2004). The culture mediums for all cell lines contained 10% fetal bovine serum (FBS; Gibco, CA, USA), 100 U/mL of penicillin, and 100 µg/mL of streptomycin. And cells were cultured at 37°C in a humidified incubator with air charge of 5% CO2.
Cell treated with 25 mM PuGNAc (Sigma, St Louis, MO, USA) and 4 mM GlcNAc (Sigma, St Louis, MO, USA) for 24 h to up-regulate O-GlcNAc level. Cells were treated with 0.1 mg/mL of Cycloheximide (Chx; Sigma, St Louis, MO, USA) for 1, 2, 4, 8, and 24 h to explore protein stability, all respectively.
Cell treated with 25 mM PuGNAc (Sigma, St Louis, MO, USA) and 4 mM GlcNAc (Sigma, St Louis, MO, USA) for 24 h to up-regulate O-GlcNAc level. Cells were treated with 0.1 mg/mL of Cycloheximide (Chx; Sigma, St Louis, MO, USA) for 1, 2, 4, 8, and 24 h to explore protein stability, all respectively.
5
Measuring Single-cell Fluorescence in Yeast
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Cells were grown at 30°C overnight in rich media lacking a sugar source. This media, YEP, contained 10 g/L yeast extract and 20 g/L Bacto-peptone. This culture was diluted to an optical density (OD600) of approximately 0.05–0.2 and grown for an additional 6 hr in YEP media ± the indicated sugar (glucose, galactose or GlcNAc, all purchased from Millipore Sigma, St. Louis, MO).
Single-cell fluorescence was measured on a LSRII analyzer (BD Biosciences). A blue (488 nm) laser was used to excite GFP and emission was detected using a 530/30 nm bandpass filter. A yellow-green laser (561 nm) was used to excite mCherry and emission was detected using a 610/20 nm bandpass filter. For each sample, 5,000–30,000 cells were measured and the mean fluorescence level was calculated. Each sample was measured three times; the mean of all three independent measurements is reported inFigures 1 , 3 and 6 . The errors in these figures refer to the standard error of the mean of these three measurements.
Single-cell fluorescence was measured on a LSRII analyzer (BD Biosciences). A blue (488 nm) laser was used to excite GFP and emission was detected using a 530/30 nm bandpass filter. A yellow-green laser (561 nm) was used to excite mCherry and emission was detected using a 610/20 nm bandpass filter. For each sample, 5,000–30,000 cells were measured and the mean fluorescence level was calculated. Each sample was measured three times; the mean of all three independent measurements is reported in
6
Metabolic Interactions of Gut Microbiome
7
Metabolic Interactions of Gut Microbiome
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P. faecium JCM 30894 and P. succinatutens JCM 16074 (JCM, Ibaragi, Japan) were grown overnight in a peptone yeast extract medium supplemented with 80 mM sodium succinate (PYS) under anaerobic conditions 19 (link). The overnight culture of Phascolarctobacterium spp. was diluted 100-fold with PYS medium. In some experiments, 100 mM GlcNAc (MilliporeSigma, St. Louis, MO) was added. For the growth assay on mucus medium, SPF WT C57BL/6 mice (some, but not all mice were littermates) were co-housed for over 2 weeks, and then randomly separated into two groups. One group was treated with αIL-22 antibody and the other with the control antibody. Colonic mucus was scraped from colonic walls into HEPES-Hank’s buffer (8.0 g/Ll NaCl, 0.4 g/l KCl, 0.05 g/l CaCl2・H2O, 0.35 g/l KH2PO4, 0.2 g/l MgSO4 • 7H2O, and 2.6 g/l HEPES, pH 7.4). Contaminating epithelial cells and membranes were removed by centrifugation, once at 12,000 g for 10 min, and once at 27,000 g for 15 min at 4°C. Colonic mucus in HEPES-Hank’s buffer was stored at −80°C until further use 56 . 300 μg of colonic mucus scrape from the control and the αIL-22 antibody–treated mice were added to the PYS medium. Phascolarctobacterium spp. were cultured anaerobically for 24–48 h. Bacterial RNAs were purified and GH family gene expression was quantified by qPCR. The CFU count was determined by culturing samples on PYS agar plates.
8
Thermal Shift Assay for Glycan Binding
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Thermal shift assays were performed using a Mini Opticon Real Time PCR machine (BioRad). 0.6 mg/mL protein in PBS was mixed with SYPRO Orange (Sigma-Aldrich, Merck, #S5692) and glycan ligand (10 mM GalNAc; Carbosynth, #MA04390; 10 mM GlcNAc, Carbosynth, #MA00834; 10 mM blood group H type-2 tetrasaccharide; Elicityl, GLY032-2) in a total reaction volume of 25 µL. The temperature was raised by 1 °C/min from 25 to 100 °C, and fluorescence readings were taken at each step.
9
Chitin Content Quantification in Mycelia
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The chitin (N-acetylglucosamine, GlcNAc) content was analyzed as follows. First, mycelial samples were freeze-dried, and then 5 mg of the dried mycelia was resuspended in 1 mL of 6% KOH and heated at 80 °C for 90 min. The samples were centrifuged (16,000× g, 10 min), and the pellets were washed with PBS over three cycles of centrifugation and resuspension (16,000× g, 10 min) before the final suspension in 0.5 mL of McIlvaine’s buffer (pH 6). An aliquot of 100 mL (13 units) of Streptomyces plicatus chitinase (Sigma, St. Louis, MO, USA) was added, and the mixture was incubated for 16 h at 37 °C with gentle mixing; 100 mL samples were then combined with 100 mL of 0.27 M sodium borate (pH 9) and heated for 10 min at 100 °C with the final addition of 1 mL of freshly diluted (1:10) Ehrlich’s reagent (10 g of p-dimethylaminobenzaldehyde in 1.25 mL of HCl and 8.75 mL of glacial acetic acid). After incubation at 37 °C for 20 min, 1 mL of the sample was transferred to a 2.5 mL plastic cuvette (Greiner, Frickenhausen, Germany), and the absorbance at 585 nm was recorded. Standard curves were prepared with GlcNAc (Sigma). The experiment was repeated three times.
10
Quantification of Reducing Sugars in Chitinase-Producing Isolates
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Isolates carrying chitinase activity were cultured separately in 10-ml tubes with 5 ml of SSP broth containing 1% SSP. After incubation for 1 to 8 days, the culture broths were centrifuged at 10,000 rpm and 4°C for 10 min, and the supernatants were collected for the colorimetric measurement of reducing sugar by the modified method of Imoto and Yagishita (
1971 (link)) with GlcNAc (Sigma-Aldrich Co., St. Louis, MO, USA) as a reference compound. Briefly, 1 ml of the color reagent was mixed with 200 μl of culture supernatant. The mixture was incubated in boiling water in an Eppendorf tube for 8 min. After cooling at room temperature, the absorbance of the mixture at 405 nm (A405) was read in a 96-well microplate using ELISA (BioTek EL800, USA). The decrease in A405 was employed to determine the reducing sugar using a standard curve.
1971 (link)) with GlcNAc (Sigma-Aldrich Co., St. Louis, MO, USA) as a reference compound. Briefly, 1 ml of the color reagent was mixed with 200 μl of culture supernatant. The mixture was incubated in boiling water in an Eppendorf tube for 8 min. After cooling at room temperature, the absorbance of the mixture at 405 nm (A405) was read in a 96-well microplate using ELISA (BioTek EL800, USA). The decrease in A405 was employed to determine the reducing sugar using a standard curve.
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