Pm150210

Mouse mononuclear macrophage RAW264.7 (CL-0190, Procell, Wuhan, China) were cultured in Dulbecco’s modified eagle medium (PM150210, Procell) with 100 U/mL penicillin and 100 U/mL streptomycin (PB180120, Procell) at 37 ℃, with 5% CO₂. The culture medium was renewed every 3 days. When cell confluence reached 80%, cell passage was performed with 0.25% trypsin/ethylenediamine tetraacetic acid.
At 24 h post cell seeding in 6-well plates, RAW264.7 cells were treated with 20 μg/mL macrophage-EV, 20 μg/mL macrophage-EV/siHMGB, or 50 μg/mL ox-LDL for 48 h, or with 1 μg/mL LPS for 24 h. Cells treated with an equal volume of PBS were taken as the control.

Rat breast cancer cell line MADB106 (CL-0148) was obtained from Procell (China) and was cultured in DMEM medium (PM150210, Procell) with 10% fetal bovine serum (FBS, 164210-500, Procell) and 1% penicillin-streptomycin solution (P/S, PB180120, Procell). SH-SY5Y human neuroblastoma cells (CL-0208, Procell, China) were cultured in MEM/F12 medium (PM151220, Procell) supplemented with 15% FBS and 1% P/S. All cells were cultured at 37 °C with 5% CO₂.

The 786-O (CL-0010), ACHN (CL-0021) human renal cancer cell lines, and 293T cells (CL-0005) were purchased from Procell Life Science & Technology (Wuhan, China). All cells were subjected to STR authentication by Procell Life Science & Technology and tested for mycoplasma contamination. The 786-O cells were cultured with RPMI-1640 (PM150110, Procell Life Science & Technology) plus 10% fetal bovine serum (FBS) (164210-500, Procell Life Science & Technology) and 1% penicillin/streptomycin (P/S) (PB180120, Procell Life Science & Technology). The ACHN cells were maintained with MEM (PM150410, Procell Life Science & Technology) supplemented with 10% FBS (164210-500, Procell Life Science & Technology) and 1% P/S (PB180120, Procell Life Science & Technology). The 293T cells were cultured in DMEM (PM150210, Procell Life Science & Technology) supplemented with 10% FBS and 1% P/S. All of these cells were maintained in 5% CO₂ at 37 °C.

To validate the expression patterns of REEP4 at the cellular level, human astrocyte (HA) cell lines and the SHG-44 of LGG cell line were selected for in vitro experiments. Cells were cultured using a complete medium formulated with 89% high glucose medium (Cat PM150210, Procell, China), 10% fetal bovine serum (FBS) (Cat 10099141, Gibco, USA), and 1% penicillin and streptomycin (Cat P1400, Solarbio, China). They were then incubated in a constant temperature incubator at 37°C with 5% CO₂. When cell fusion approached 100%, the cells were passaged at 50% and cultured for subsequent experiments. For the methylation assay, SHG-44 cells were treated with 100 uM of ademetionine disulfate tosylate (SAM) when their cell fusion approached 70% (Cat 97540-22-2, Topscience, China) for 10 h. The treated cells were harvested to identify the REEP4 RNA level.

Human primary podocytes were purchased from Huatuobo (HTX2426, OTWO, Shenzhen, China) and cultured in RPMI1640 (PM150110, Procell, Wuhan, China) supplemented with 20 µ/ml γ-interferon, 10% foetal bovine serum (164210-500, Procell), and 1% penicillin/streptomycin (PB180120, Procell) at 33°C in a 5% CO₂-containing environment. The cells were further cultured at 37°C for 10 days to induce podocyte maturation.
HMCs derived from human glomerular mesangial cells were purchased from Procell (CL-0619) and cultured in Dulbecco’s Modified Eagle Medium (Procell, PM150210) supplemented with 10% foetal bovine serum (164210-500, Procell, China) and 1% penicillin/streptomycin (PB180120, Procell). The cells were subcultured at 37°C in a 5% CO₂ atmosphere.

MDA-MB-231 and SUM-149 were purchased from the American Type Culture Collection (ATCC, USA), and MDA-MB-231 was cultured in high glucose DMEM medium (PM150210, Procell) supplemented with 10% fetal bovine serum (164210, Procell) and 1% penicillin and streptomycin (PB180120, Procell). SUM-149 was cultured in Ham’s F12 medium (L410KJ, BasalMedia) supplemented with 5% fetal bovine serum, 5µg/ml of insulin (UNITED LABORATORIES), and 1µg/ml of hydrocortisone (R011855, RHAWN). MDA-MB-231-Re epirubicin-resistant cell lines were established as previously described (11 (link)). All cells were cultured in 5% CO₂, at 37°C.

Normal rat renal TEC line NRK-52E is from Center for Excellence in Molecular and Cellular Sciences, Chinese Academy of Sciences (China), routinely cultured with DMEM (#PM150210, Procell, China) containing 5% fetal bovine serum in incubator of 37°C, 5% carbon dioxide. To evaluate the role of FPS-ZM1 in cisplatin-induced cellular insult, NRK-52E cells were pretreated with FPS-ZM1 alone for 6 h, followed by co-treatment with cisplatin for 24 h.