Ag490

ESCs were treated with the following reagents in the described experimental procedures: 100 ng ml⁻¹ Dkk1 (R&D, cat. 5897-DK); 100 ng ml⁻¹ Sfrp1 (R&D, cat. 1384-SF); 100 ng ml⁻¹ Wnt-3a (R&D, cat. 5036-WN); 12 ng ml⁻¹ bFGF (Invitrogen, cat. 13256029); 20 ng ml⁻¹ Activin-A (R&D, cat. 338-AC); 3 μM CHIR99021 (AbCam, cat. ab120890); 1 μM PD 0325901 (Sigma, cat. N°PZ0162); 1 μM EPZ005687 (BioVision, cat. 2364); 2 μM GSK126 (BioVision, cat. 2282); 5 μM AG490 (Invivogen, cat. N°tlrl-ag4); 1 μM Jak1 (Millipore, cat. N°420099); 3 μM XAV939 (AbCam, cat. N°ab120897).

Human SGC-7901 and MGC-803 cells, normal gastric epithelial cells (GES-1) were purchased from the Cell Bank of Chinese Academy of Medical Science (Shanghai, China). All cells were cultured in RPMI-1640 medium supplied with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37 °C in a humidified atmosphere of 5% CO₂. Cells were routinely tested for mycoplasma contamination (MycoAlert™ PLUS Mycoplasma Detection Kit, Lonza). For cell culture, 50–60% confluent cells were transiently incubated with specific agents for 48 h until the extraction of RNA and protein lysates.
Purified recombinant human C3 protein (HuC3, 20 ng/ml, MBS230377, MyBioSource, San Diego, CA) was added to or left out of the culturing medium. Exogenous C3 was depleted with cobra venom factor (CVF; Heng Fei biological technology, Shanghai, China), as previously described [20 (link)]. Complement receptor 1 (CR1/CD35, MBS717740, MyBioSource) was used to block C3 activation as previously confirmed [21 (link)], with JAK2 blocker (AG490, 25 μM, InvivoGen, Hongkong) used to inhibit STAT3 signaling pathway [22 (link)].