Tris hcl

Compounds, such as NSC13626 were requested from National Institute of Health (Bethesda, MA, United States). HCT116 CRC line was obtained from Bioresource Collection and Research Center (Hsinchu, Taiwan). McCoy’s 5A cell culture medium, sulforhodamine B (SRB), dimethyl sulfoxide, ethylenediaminetetraacetate, propidium iodide, and Triton X-100 purchased from Sigma (St. Louis, MO, United States). Trichloroacetic acid (TCA), acetic acid, Trizma base, Tris–HCl, sodium chloride, sodium dodecyl sulfate, ethanol, citric acid, and Na₂HPO₄ were purchased from Wako Pure Chemical Industries (Osaka, Japan). Cocktail protease inhibitor was obtained from Calbiochem Research Biochemicals (Merck, Burlington, MA, United States). RNase A was purchased from Bioshop (Ontario, Canada).

Bovine serum albumin (BSA) (FUJIFILM Wako Pure Chemical Corporation) was prepared by dissolving in 0.1 mol/l Tris-HCl (FUJIFILM Wako Pure Chemical Corporation) (pH 8.0) to a concentration of 5% and filtered through a 0.22-µm filter for sterilization. Oleic acid (FUJIFILM Wako Pure Chemical Corporation) was dissolved in 5% BSA solution to prepare 4 mM Oleic acid solution. Oleic acid use solution was diluted to 0, 50, 200 and 500 µM with the aforementioned culture medium.

Fecal DNA was extracted based on a previous report with some modifications [57 (link)]. Approximately 0.2 g of fecal sample was suspended in a 50-mL Falcon tube containing 20 mL of PBS. The suspension was washed with 10 mL of PBS and debris was removed with a 100-µm mesh nylon filter (Corning Inc., New York, NY, USA). After centrifuging the filter at 4000 rpm for 20 min at 4 °C, each precipitate was suspended in 1.5 mL of TE10 buffer composed of 10 mM Tris-HCl (FUJIFILM Wako Pure Chemicals Co., Ltd.) and 10 mM EDTA (Dojindo, Tokyo, Japan). After the suspension was transferred to another microtube and centrifuged at 10,000 rpm for 5 min at 4 °C, each precipitate was resuspended in 0.8 mL of TE10 buffer. DNA was extracted using 1 mL of PCI (Invitrogen, Carlsbad, CA, USA), and 0.1 mL of lysozyme (FUJIFILM Wako Pure Chemicals Co., Ltd.) and 0.2 mL of achromopeptidase (FUJIFILM Wako Pure Chemicals Co., Ltd.) was used to isolate the DNA. The DNA was treated with RNase (Promega Corp., Madison, WI, USA) and then purified by precipitation with 20% polyethylene glycol (PEG) solution (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). The DNA was rinsed with 70% ethanol and dissolved in 50 μL of TE buffer.

Pg-OMVs concurrently carry many different proteins (enzyme gingipains and fimbrial proteins), which are derived from the bacterial outer and periplasmic space [37 (link)]. For this reason, they were purified and activated with cysteine according to the method previously described by Kariu et al. [38 (link)] The activated proteases were diluted with 0.1 M Tris-HCl (pH 7.6) buffer (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) containing 50 mM NaCl (Fujifilm Wako Pure Chemical Corporation) and 5 mM CaCl₂ (Nacalai Tesque, Kyoto, Japan) directly before assays. Phosphorylated dihydroceramide was isolated as described in the previous literature [42 (link)]. Purity of the lipid isolate was confirmed by liquid chromatography–mass spectrometry, and its structure verified prior to Raman analyses by means of electrospray ionization (ESI) MS/MS (as described in Ref. [42 (link)]). For biological experiments, PGDHC was dissolved in 70% ethanol.

RPMI 1640 medium was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Opti-modified Eagle’s medium (Opti-MEM) and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Sodium chloride, sodium bicarbonate, potassium chloride, Tris-HCl, ethylenediaminetetraacetic acid (EDTA), ammonium chloride, and glucose were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Monothioglycerol, MEM non-essential amino acids, and penicillin-streptomycin-glutamine mixed solution were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). The 20 bp ladder was purchased from Takara Bio (Otsu, Japan). Propidium iodide, R848, and ovalbumin were obtained from Merck KGaA (Darmstadt, Germany). Alexa Fluor 488 annexin V/ Dead Cell Apoptosis kit with Alexa Fluor 488 annexin V and PI for Flow Cytometry was purchased from Invitrogen (San Diego, CA, USA). All other chemicals were of the highest grade available and were used without further purification.

The dose response range of partially purified TgMQO was assayed at 37 °C using a UV760 (Jasco-Japan, Tokyo, Japan) at different concentrations of purified TgMQO, ranging from 0.025 to 5 µg/mL, in 1 mL of the reaction mixture as previously described [23 (link)] in triplicate, with a minor change of KCN replaced with 50 nM AF.
Optimization of temperature for the TgMQO assay was performed spectrophotometrically using a UV760 (Jasco-Japan) equipped with a water bath circulator (Taitec). The TgMQO activity was assayed using a 1 mL black quartz cuvette with 50 mM HEPES pH 7.0, 120 μM DCIP, 50 nM AF, 20 μM dUQ, and 1.5 μg/mL purified TgMQO at varying temperatures in triplicate. The reaction was started by the addition of 10 mM malate, and the activity was calculated as described previously [23 (link)].
The optimum pH was determined by measuring TgMQO activity at different pHs using 50 mM of sodium phosphate (NaPi, pH 5.8–8.0), potassium phosphate (KPi, pH 5.8–8.0), HEPES-NaOH (6.8–8.4), Tris-HCl (Wako) (pH 6.9–9.0), MOPS-NaOH (Dojindo) (pH 6.5–9.0), or CHES-NaOH (Dojindo) (pH 8.6–10.0) buffers at 37 °C with a SpectraMax^® Paradigm^® Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA), as described previously [23 (link)].