Rpmi 1640 medium

Human esophageal squamous cell carcinoma (ESCC) cell lines KYSE150 and KYSE510 were generously provided by Dr. Xu Liyan at Shantou University Medical College. The cells previously described [21 (link),22 (link)] were maintained at 37 °C with 5% CO₂ in RPMI1640 medium (Biosharp Life sciences, Beijing, China) supplemented with 10% FBS (TransGen, Beijing, China). For EGCG stimulation assays, we inoculated the cells at a concentration of 1.6 × 10⁵/mL in full-serum medium and changed to RPMI1640 medium without FBS 24 h later, then treated the cells with EGCG at indicated concentrations. We also performed EGCG stimulation without changing the medium. Inhibition of p65 was performed by treating the cells with 5 μM BAY11-7082 (Beyotime Biotechnology, SF0011, Shanghai, China) for 1~3 h.

The femur and tibia medullary cavity of C57/BL6 mice were rinsed with Roswell Park Memorial Institute (RPMI)-1640 medium (Biosharp, Hefei, China) to obtain primary cells. Neutrophils were isolated using the EasySep mouse neutrophil enrichment kit (Stemcell Technologies, Vancouver, BC, Canada) according to manufacturer instructions and cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA) and 0.1 mg/mL primocin (InvivoGen, San Diego, CA, USA).
EA.hy926, L929, and RAW264.7 cells were incubated in high-glucose Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.

The human non-small cell lung cancer (NSCLC) cell lines H522, A549, H1299, H460, the human bronchial epithelium cell line (BEAS-2B),human monocytes cell line THP-1 and mouse Lewis lung carcinoma cell line (LLC), were all obtained from the American Type Culture Collection (ATCC, Manassas, VA). These cell lines were all grown in a controlled environment at 37 °C with a 5% CO₂ concentration, using RPMI 1640 medium (Biosharp, Guangzhou, China) or DMEM medium (Biosharp, Guangzhou, China). The media were further supplemented with 10% fetal bovine serum (FBS; Gibco, South America).

The BxPC-3 cells were cultured in RPMI 1640 medium (Biosharp, Hefei, China) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Cromwell, CT, USA), sodium pyruvate (1 mM), streptomycin (100 μg/mL), and penicillin (100 units/mL) (Biosharp) at 37° C in 5% CO².
Total RNA was isolated from BxPC3 cells. The designed primers were used to amplify decorin by RT-PCR, followed by the construction of the expression vector pcDNA3.1-DNC. The Xfect Transfection reagent (Takara, Osaka, Japan) was used for transfection. PC cells (1 × 10⁵ cells/well) were plated in a 6-well plate, and 12 hours later, Xfect polymer was incubated with pcDNA3.1-DNA for 10 min and the mixture was added dropwise to the respective wells. After 4 h incubation, the serum-containing medium was added. The pcDNA3.1 empty vector was used as negative control.

Bone marrow was flushed from femur and tibia bones using 10 ml syringe with 21 G needles and cells were cultured in RPMI 1640 medium (Bio‐sharp, China) with 10% FBS (10099141, Gibco), penicillin/streptomycin (100 μg/ml) and 10% (vol/vol) conditional medium of L929 mouse fibroblasts for 7 days.⁶⁰, ⁶¹ For M1 Mφs activation, cells were incubated for 48 h with 100 ng/ml LPS (Solarbio, China) and for M2 Mφs activation, cells were incubated for 48 h with 20 ng/ml IL‐4 (Peprotech, USA). The efficiency of polarisation was checked by flow cytometry analysis with 98.9% in M1 induction and 98.2% in M2 induction, respectively. For L929 conditional medium collection, L929 cells were cultured in RPMI 1640 containing with 10% FBS and 1% penicillin/streptomycin. Cells should become confluent in 2–3 days and supernatants were collected 2 days later.

HTR-8/SVneo cell line, an extravillous trophoblastic cell line, was obtained from Dr. Charles Graham (Queen’s University, Canada) as a gift. Cells were cultured in RPMI 1640 medium (Biosharp, China) supplemented with 10% fetal bovine serum (Gibco, USA) and 100 U/mL penicillin & streptomycin (PYG0016, Boster, China) at 37 °C in an incubator containing 5% CO₂. HTR-8/SVneo cells were cultured in a 6-well plate and transfected with Lipofectamine 3000 (Invitrogen, USA). A TMBIM6 overexpression model was established via TMBIM6 plasmid transfection for 24 h. Simultaneously, a negative control model was established via an empty vector transfection. Knockdown of METTL3 and YTHDF2 was achieved using three shRNAs targeting the METTL3 and YTHDF2 genes, which were synthesized by GenePharma Biotech. The target sequences are listed in Additional file 2: Table S2. Transfection efficiency was confirmed by qRT-PCR or WB. To construct a model of ER stress, HTR-8/SVneo cells were incubated with 100 nmol/L TG (T863962; Macklin Biochemical Co., Ltd, China) for 6 h after transfection with overexpression plasmids or shRNAs and incubated for the last 18 h. Finally, HTR-8/SVneo cells were collected to extract the RNAs and proteins for subsequent experiments.