Dulbecco modified eagle medium (dmem)

Two human pancreatic cancer cell lines (PANC-1 and SW1990) and a normal pancreatic duct epithelia cell line (HPDE6-C7) were purchased from Procell Life Science&Technology (Wuhan, China). PANC-1 cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium) containing 10% FBS (fetal bovine serum) and 1% P/S (penicillin/streptomycin) (Procell, Wuhan, China). SW1990 cells were cultured in Leibovitz’s L-15 medium containing 10% FBS and 1% P/S (Procell, Wuhan, China). HPDE6-C7 cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium) containing 10% FBS and 1% P/S (Procell, Wuhan, China). The culture conditions were 37°C with 5% CO₂.
SLC1A5-specific shRNA and amplification plasmids were designed by Hanheng Biotechnology (Shanghai, China). Lentiviruses were also purchased from Hanheng Biotechnology (Shanghai, China) and were applied to transfect cells. The interference efficiency was tested by RT-qPCR after 72 h post-transfection.

Cell migration and invasion were investigated by transwell chambers (Millipore). Specially, the chambers were pre‐incubated with Matrigel (Corning) to reveal cell invasive ability. Briefly, cells were homogenized in serum‐free DMEM (Procell) and placed into the upper chambers. DMEM containing 15% FBS (Procell) was added into the lower chambers. After 24 h, the cells were incubated with methanol (Beyotime) and crystal violet (Beyotime), respectively. Results were confirmed by figuring up the number of cells in the lower chambers with an Eclipse Ts2R inverted microscope (Nikon) at a × 100 magnification.

RAW264.7 cells were obtained from ATCC and cultured in DMEM (Procell, Wuhan, China) containing 10% fetal bovine serum (Procell) in an incubator at 37°C and 5% CO2.
RAW264.7 cells were seeded into 6-well plates the day before transfection at a confluency of 70%–80% for transfection experiments. After the cells had grown for 24 h, the medium was changed to serum-free medium. Then, 200 μL of DMEM (Procell) was incubated at room temperature for 20 min with 100 pmol of siAhR pool or siNC (GenePharma, Shanghai, China) and 8 μL of Lipofectamine 2000 (Invitrogen, California, United States) to facilitate complex formation. Subsequently, the cells were cultivated for 48 h at 37°C and 5% CO₂ in an incubator. The transfection mixture was gradually added to each well. The cells were then grown in an incubator for 48 h at 37°C with 5% CO₂.
The cells were divided into six groups: control, LPS, LPS + NAR, LPS + NAR + siAhR, LPS + NAR + siNC, and LPS + NAR + CH223191. The dosages administered were 1 μg/mL for LPS, 1 μM for CH223191 (Wang et al., 2018 (link)), and 50 μM for naringenin. CH223191 was added 30 min after naringenin was added. The treatments were conducted 24 h later.