Bicinchoninic acid (bca)

BGC-823 and SGC-7901 cells (~5×10⁵) were seeded in a 6-well culture plate at 50%–60% confluence. Then, 2 μg exosomes (equivalent to those collected from ~5×10⁶ BM-MSCs) per well, based on the protein concentration determined by a bicinchoninic assay (BCA; Sangon Biotech), were added to GC cells in the 6-well plate. After cultivation for 24 hours, exosomes were resuspended in PBS and added to the cells at a final concentration of 200 μg/mL. The expression level of miR-221 in the cancer cells was analyzed 12 hours later by qRT-PCR.

The purified microglia cells were cultured in 6-well plates at 1 × 10⁶ cells. The supernatant is collected and centrifuged to remove the precipitate for testing secretion of inflammatory cytokines. BCA (Sangon Biotech, Sichuan, China) was used to determine the concentration of total protein, diluted to the same concentration, and then operated in strict accordance with the instructions of the ELISA Kit (BOSTER, Wuhan, China) to determine the levels of IL-10, IL-1β and TNF-α.

After transfection of BMMSCs with FAPα-siRNA, BMMSCs (2×10⁵) and Kasumi-1 cells (5×10⁵) were cocultured in 6-well plates. After 48 h, Kasumi-1 cells were collected and washed twice with PBS. Total cell lysates were obtained using lysis buffer (Beyotime Institute of Biotechnology) supplemented with 1 mM PMSF. The protein concentration was determined using the BCA (Sangon Biotech Co., Ltd.) method according to the manufacturer's guidelines. An equal amount of protein (20-40 g) was separated via 10-12% SDS-PAGE and transferred to PVDF membranes. Next, membranes were blocked with 5% non-fat milk for 2 h at room temperature. PVDF membranes were incubated with anti-actin and anti-β-catenin antibodies (cat. no. 2698S; Cell Signaling Technology, Inc.; 1:500) overnight at 4°C. Subsequently, membranes were washed and incubated with a horseradish peroxidase-conjugated antibody (cat. no. 7074S; Cell Signaling Technology, Inc.; 1:5,000) in 0.2% TBS-Tween at room temperature for 2 h. After being washed, protein bands were visualized using an ECL detection kit (Biological Industries). Semi-quantification of the western blotting was performed using the Bio-Rad imaging system and ImageJ software (29 ) (Life-Line Fiji versions java8; http://imagej.net/Fiji/Downloads) to detect protein expression.