Acetate

For in vitro studies, MZ-CRC-1 cells were seeded in a 6-well plate in a volume of 2 mL cell suspension medium so that 1 × 10⁶ cells were entered into each well and cultured overnight. The medium was replaced with serum-free medium the next day, followed by treatment with 250 μM acetate (Sigma-Aldrich, St. Louis, MO, USA). Culture medium was collected at 0, 1, 3, 5, and 10 min after acetate administration, and calcitonin levels were measured using a commercial ELISA Kit for Calcitonin (CEA472Hu; Cloud-Clone Corporation, Houston, TX, USA), as per manufacturer’s instructions.

EBC samples collected for a previous study^{53 (link)} were utilized. The details of the sample collection procedure have been previously described, but briefly, 1–2 mL of EBC was collected during 20 min of tidal breathing from each of seven adult subjects using an EcoScreen device (Jaeger, Wurzburg, Germany), which condensed the exhaled breath at −20 °C. All surfaces were triple-rinsed with nitrite-free water prior to contacting the EBC, and the samples were frozen at −80 °C for later analysis. In the previous study, nitrite concentrations were measured using selective catalytic reduction and chemiluminescence detection (NOA 280i, GE Analytics, Boulder, CO, USA)^{53 (link)}. All solutions were prepared with distilled water. For testing and calibration of the sensors, we experimented with various buffers, including sodium nitrite, acetate, and phosphate buffers (Sigma-Aldrich, St Louis, MO, USA).

For Treg/Teff cell staining (Yang, et al., 2018 (link)), live/dead cells were first labeled with antibodies to distinguish living cells (Waltham, Ma, United States). The antibodies used included; anti mouse CD4 percp-cy5.5, anti-mouse cd45-bv510, anti-mouse IL-17A APC and anti-mouse IFN-γ PE, anti-mouse TNF-α Bv421, anti-mouse Foxp3 FITC (all from BioLegend). For Teff cells, cytokine staining analysis mainly containing Th17 and Th1, the cells mixed with acetate (50 ng/ml; Sigma-Aldrich), ionomycin (500 ng/ml; Sigma-Aldrich) and brefidine (1 µg/ml; Sigma-Aldrich) were cultured together and placed in 96 well plates with cytokine secretion blockers for 4 h. Stained cells were measured using BD LSR Fortessa flow cytometer (BD Biosciences) and data were obtained and were analyzed with Flowjo 10.0 (Flowjo company, United States).

ECP was used and prepared as previously described [1 (link),2 (link),3 (link)]. ECP was mainly composed of rhamnose (49.7%), glucose (29.9%), glucuronic acid (10.8%), and xylose (7.2%). In addition, small amounts of mannose (1.0%) and galactose (1.3%) were also detected. The molecular weight of ECP was 11.67 kDa, and the sulfate content of ECP was 14.7%. Bacterial nutrients used for the in vitro anaerobic fermentation were all obtained from Sigma-Aldrich (St. Louis, MO, USA). The standard SCFA solutions, including lactate, acetate, propionate, and butyrate, were also obtained from Sigma-Aldrich (St. Louis, MO, USA). Hemin and L-cysteine hydrochloride used for the fermentation were purchased from Sangon Biotech (Shanghai, China). All other analytical-grade chemicals were acquired from Sinopharm Chemical (Shanghai, China).

MDA-MB-231 cells were seeded in the upper chamber of Corning Costar Transwell cell culture inserts (Merck, Darmstadt, Germany) coated with 20 μL 25% Matrigel (R&D Systems) at 1 × 10⁵ cells in 100 μL of serum-free medium. The lower chamber was filled with 600 μL DMEM containing 10% FBS. After 48 h of incubation, MDA-MB-231 cells were fixed with a fixing solution containing 75% methanol and 25% acetate (Sigma) for 10 min, and cells on the upper side of the membrane surface were removed by scraping with a cotton swab. The cells that passed through the filter were stained with 0.1% crystal violet (Sigma) for 10 min. The cell migration and invasion ability of the stained cells were captured by microphotograph and measured using ImageJ software.