In a series of experiments each aneurysm tissue sections were incubated in medium in the presence and absence of DL-homocysteine. Aneurysm fragments were cut into approximately 2 mm pieces. Equal wet weights of the tissue were placed in each well of 12-well plates with serum-free Dulbecco's Modified Eagle's Medium (DMEM) with 4500 mg/L glucose (without phenol red and L-glutamine; Sigma-Aldrich, St. Louis, Missouri, USA). The thick and thin ILTs and adjacent walls were separately incubated without or with 100 μmol/L or 500 μmol/L DL-homocysteine (DL-homocysteine ≥ 95%, Sigma-Aldrich) at 37°C for 6 h in humidified air with 5% v/v CO2. From the wide range of DL-Hcy concentrations mentioned in the literature (10-2000 μmol/L) the two values were selected (as discussed). The incubation times were chosen based on previous studies [29 (link)]. Nontreated aneurysm tissues were used in each experiment as controls. After treatment, tissue samples were collected for protein extraction.
Dl homocysteine
Manufactured by Merck Group
Sourced in United States, Germany
DL-homocysteine is a chemical compound used in laboratory settings. It serves as a reference material for analytical procedures involving the detection and quantification of homocysteine, an amino acid that is commonly measured for various clinical and research applications.
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Lab products found in correlation
L methionine, by Merck Group (5 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (4 mentions)
Choline chloride, by Merck Group (3 mentions)
Adenosine, by Merck Group (3 mentions)
Streptomycin, by Corning (3 mentions)
L cysteine, by Thermo Fisher Scientific (3 mentions)
L cysteine, by Merck Group (3 mentions)
Methionine, by Merck Group (3 mentions)
Thapsigargin, by Merck Group (3 mentions)
Tunicamycin, by Merck Group (3 mentions)
Oleic acid, by Thermo Fisher Scientific (2 mentions)
Sodium homogamma linolenate, by Nu-Chek Prep (2 mentions)
D glucose, by Thermo Fisher Scientific (2 mentions)
L methionine, by Thermo Fisher Scientific (2 mentions)
Methylcobalamin, by Merck Group (2 mentions)
Zileuton, by Merck Group (2 mentions)
Hygromycin b, by Thermo Fisher Scientific (2 mentions)
Erythro 9 2 hydroxy 3 nonyl adenine hydrochloride, by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Dialyzed fbs, by Gemini Bio (2 mentions)
42 protocols using dl homocysteine
1
DL-Homocysteine's Effect on Aneurysm Tissue
2
Nutrient Supplementation for Paralysis Assays
3
Nutrient Supplementation for Paralysis Assays
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Nutrient supplemented plates for paralysis assays contained 10mM D(+)-glucose (Fisher Scientific), 0.3mM sodium homogamma linolenate (Nu-Chek Prep), 0.1mM or 0.3mM oleic acid (ThermoFisher Scientific),148nM or 740 nM methylcobalamin (Millipore Sigma), 1.34 mM, 6.7 mM, 13.4mM, or 67 mM L-methionine (Fisher Scientific), 1 mM, 10 mM, or 20 mM choline chloride (Millipore Sigma), and 15mM or 30 mM DL-Homocysteine (Millipore Sigma). Supplements were added to autoclaved NGM media at 55°C. Specific concentrations used for each supplementation experiment are indicated in the figures and legends. Some of these concentrations for glucose (Alcántar-Fernández et al., 2018 (link)), fatty acids (Deline et al., 2013 ), methylcobalamin (Revtovich et al., 2019 (link)), L-methionine (Wei and Ruvkun, 2020 (link)), homocysteine (Wei and Ruvkun, 2020 (link)), and choline (Walker et al., 2011 (link)) were used in prior C. elegans work; additional concentrations were used to determine dose-dependent effects for certain treatments. Plates were stored at 4°C. Fatty acid supplemented plates were covered with foil to prevent light oxidation. Plates were seeded with the different E. coli cultures (OD600 = 1.0) and dried for three days before use.
4
Emodin Regulates Inflammatory Response
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Emodin, isolated from the root of Rheum officinale Baill, was provided by Shaanxi Zhongxin Biotechnology Co. Ltd (Xi’an, China) and dissolved in dimethyl sulfoxide (DMSO) for use. DL-homocysteine and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma–Aldrich (St. Louis, MO, USA). Polyclonal anti-rat CRP and anti-rat PPARγ antibodies were from Abcam (Cambridge, UK). Antibodies against phospho-ERK1/2, ERK1/2, phospho-p38, p38 and 2’,7’-dichlorodihydrofluorescein diacetate (H2DCF-DA) were from Beyotime (Jiangsu, China). ELISA kit for detecting CRP was from Westtang (Shanghai, China).
5
Neuroblastoma Cell Line for Tau Aggregation
6
Methionine-Deprived Resistant MDA-MB-468 Cell Lines
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MDA-MB-468 were maintained in DMEM (Sigma-Aldrich, D0422) supplemented with 10 % dialyzed FBS (Gemini Bio-Products), 1.5 μM cyanocobalmin (vitamin B12), 4 mM l -glutamine, 100 μM l -cysteine (Fisher Scientific), and 100 μM l -methionine (Sigma-Aldrich). In the case of methionine-free media, 370 μM DL-homocysteine (Sigma-Aldrich) or 370 μM DL-homocysteine-2H4 (13C Molecular, 12714-158) was added in the absence of methionine.
Resistant cell lines were isolated as described in Hoffman et al. [17 (link)]. Briefly, MDA-MB-468 resistant clones were isolated after prolonged culturing in methionine-free media. The majority of MDA-MB-468 cells detach; however, resistant clones begin to appear after 2 weeks. Clones were isolated, and proliferation rates were measured using CellTiter-Glo luminescent cell viability assay (Promega).
Both MD468 and MB468res-R8 cell lines were tested for authentication via STR profiling in January 2016 by Genetica DNA Laboratories (a LabCorp brand; Burlington, NC) using the commercially available PowerPlex® 16HS amplification kit (Promega Corporation) and GeneMapper ID v3.2.1 software (Applied Biosystems). Authentication of each cell line was confirmed by a 100 % match to the reference STR profile of MDA-MB-468 (ATCC HTB-132) cells from ATCC.
Resistant cell lines were isolated as described in Hoffman et al. [17 (link)]. Briefly, MDA-MB-468 resistant clones were isolated after prolonged culturing in methionine-free media. The majority of MDA-MB-468 cells detach; however, resistant clones begin to appear after 2 weeks. Clones were isolated, and proliferation rates were measured using CellTiter-Glo luminescent cell viability assay (Promega).
Both MD468 and MB468res-R8 cell lines were tested for authentication via STR profiling in January 2016 by Genetica DNA Laboratories (a LabCorp brand; Burlington, NC) using the commercially available PowerPlex® 16HS amplification kit (Promega Corporation) and GeneMapper ID v3.2.1 software (Applied Biosystems). Authentication of each cell line was confirmed by a 100 % match to the reference STR profile of MDA-MB-468 (ATCC HTB-132) cells from ATCC.
7
Homocysteine Metabolism Regulation
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l -Methionine, d,l -homocysteine, vitamin B12, and folate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit monoclonal anti-p-eIF2α and rabbit polyclonal anti-β-Tubulin were purchased from Cell Signaling Technologies (Danvers, MA, USA). Rabbit polyclonal anti-phospho- IRE1α was purchased from Novus Biologicals (Littleton, CO, USA). Rabbit polyclonal anti-t-IRE1α, rabbit polyclonal anti-GRP78, mouse monoclonal anti-CHOP, and rabbit polyclonal anti-homocysteine were purchased from Abcam (Cambridge, MA, USA). Annexin V-FITC Kit was purchased from MBL (Nagoya, Japan).
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8
Biochemicals for Molecular Research
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DL‐homocysteine (44 925), DTT (43 815), NaHS (161 527) and GYY4137 (SML0100) were purchased from Sigma‐Aldrich (USA).
9
Quantifying Cellular Fitness in Co-Cultures
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Co-cultures were generated by mixing equal proportions (1 OD600) of cells expressing fluorescent GFP and non-fluorescent GFP-Y66F in 10 mL of synthetic complete media. Immediately following mixing, approximately 0.15 optical densities were collected, washed, resuspended in 2 mL of sodium citrate buffer (50 mM sodium citrate, 0.02% NaN3, pH 7.4), and stored at 4°C for subsequent flow cytometric analysis. All co-cultures were then diluted to 0.004–0.01 optical densities in 10 mL of synthetic complete media with or without stress (0.2 M CaCl2 or 0.4 M KCl) and incubated at 30°C with continuous rolling. Where indicated, media was supplemented with 250 μM reduced L-glutathione (Sigma), 250 μM DL-homocysteine (Sigma), or 5 mM L-ascorbic acid (Sigma). Co-cultures were sampled and diluted to 0.004–0.02 optical densities, as described above, every 12 hours to ensure that the population did not exceed logarithmic growth during the duration of the experiment. Following sonication, the percentage of GFP and GFP (Y66F) at each timepoint was quantified using a Guava EasyCyte HT flow cytometer and analyzed with FlowJo software. Replicates in which a fitness difference was observed between the GFP and GFP (Y66F) controls were excluded from the final analysis.
10
Progranulin Modulates HUVEC and THP-1 Interaction
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Human umbilical vein endothelial cells (HUVECs) were purchased from Sciencell Research Lab. HUVECs were cultured in endothelial cell medium (ECM) media containing 5% fetal bovine serum (FBS), 1% endothelial cell growth additive (ECGs), and 1% penicillin/streptomycin solution. THP-1 monocytes were cultured in RPMI-1640 medium (Sigma R8758) supplemented with 10% FBS. The cells were placed in an incubator at 37°C with a humidified atmosphere of 5% CO2. The cells were stimulated with DL-homocysteine (Sigma Aldrich) 2.0 mM in the presence or absence of recombinant human progranulin (rhPGRN) protein 200 ng/mL (R&D systems) for 24 h as well as treated with a variety of concentrations of rhPGRN (0, 50, 100, 200, 400 ng/mL) for 24 h depending on the experimental goals. The CCK8 kit and BCA protein assay kit were obtained from Beyotime (Beyotime Biotech, Shanghai, China). Granulin, VCAM1 antibodies were purchased from Abcam (Cambridge, United Kingdom), and VE-cadherin, p-EphA2 (Ser897), EphA2, p-AKT (Ser473), AKT, p-NF-κB p65 (Ser536), and NF-κB p65 antibodies were obtained from Cell Signaling Technology (Danvers, MA, United States).
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