F12k medium

Cell lines HepG2.215, PLC5, HepG2, LO2, AML12, and FL83B were all purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai). HepG2.215 and PLC5 are both HBV-positive (termed as HBV + hereafter) HCC cell lines. HepG2.215 is a stably transfected with HBV, while PLC5 only secretes HBsAg but does not support HBV replication. HepG2 is an HBV-negative (termed as HBV- hereafter) HCC cell line. LO2, AML12, and FL83B are normal immortalized hepatocytes. LO2 is human origin while AML12 and FL83B are mouse origin.
HepG2, HepG2.215, PLC5 and LO2 were cultured in high glucose DMEM (Sangon) supplemented with 10% FBS (Hyclone) and penicillin (100 U/ml) and streptomycin (100 µg/ml) (Sangon). AML12 cells were cultured in DMEM/F12 medium (Sangon) supplemented with 10% FBS, 10 µg/mL insulin (ThermoFisher Scientific), 5.5 µg/mL transferrin (ThermoFisher Scientific), 5 ng/mL sodium selenite (ThermoFisher Scientific), 40 ng/mL (100 nM) dexamethasone (ThermoFisher Scientific) and antibiotics. FL83B cells were cultured in F-12K medium (Procell) supplemented with 10% FBS and antibiotics. Brassicasterol (cat no: B4936) was purchased from Sigma-Aldrich, Merck and sorafenib (cat no: sc-220125) was purchased from Santa Cruz and AKT agonist IGF-1 (cat no: ab9573) was purchased from Abcam.

VSMCs were diluted in F12K medium (Procell, Wuhan, China) and grown in 96-well plates for 16h. Then, cells were incubated with various concentrations (0, 25, 50, 75 and 100g/mL) of ox-LDL (Yeasen, Shanghai, China) for 24h or 50g/mL ox-LDL for different time (0, 12, 24, 36 and 48h). After that, MTT reagent (Solarbio, Beijing, China) was incubated with cells for 4h. Cell supernatant was discarded and dimethyl sulfoxide (Sigma, St. Louis, MO, USA) was used to dissolve formazan. The cell viability was determined by assessing the output of wavelength at 490 nm with a Varioskan LUX Multimode microplate reader (Thermo Fisher, Waltham, MA, USA).

VSMCs were seeded in 6-well plates for 16h and treated with 50g/mL ox-LDL (Yeasen, Shanghai, China). Twenty-four hours later, si-MIAT, miR-641 inhibitor, miR-641 mimic or pcDNA-STIM1 was transfected into the cells at ~70% confluence with controls according to the defined purposes. Then, cells were cultured for 2 weeks. F12K medium (Procell, Wuhan, China) was renewed every 3 days during culture. The forming colonies were immobilized with paraformaldehyde (Sigma, St. Louis, MO, USA) and then dyed with crystal violet (Sigma, St. Louis, MO, USA). Cell colony-forming ability was determined by assessing the number of colonies. A colony was deemed when cell numbers over 50.

Human normal bladder epithelial cell line SV-HUC-1 was purchased from ATCC. The human urinary bladder transitional cell carcinoma cell lines T24, RT4, UMUC3 and 5637 were purchased from ATCC. EJ cells were obtained from the Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences (Shanghai, China). CDDP-resistant cell lines (EJ-CDDP) were derived from our previous studies [15 (link)]. All cells were maintained in a humidified incubator with 5% CO₂ at 37℃. The SV-HUC-1 cells were cultured in F12K medium (Procell, China). The T24, EJ, RT4, 5637 and EJ-CDDP cells were cultured in RPMI 1640 medium (Procell, China). The UMUC3 cells were cultured in DMEM (Procell, China). All medium was supplemented with 10% FBS (Vazyme, China) and 1% penicillin/streptomycin (Procell, China). All cell lines were confirmed negative for Mycoplasma contamination. Cisplatin (CDDP) and actinomycin D (ActD) were purchased from Sigma-Aldrich (USA) and solubilized in DMSO.

All the human prostate cancer cell lines including PC3, DU145, LNCaP, and benign prostate hyperplasia cell line BPH-1, and human embryonic kidney cells (293T) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). According to the instructions, PC3 was cultured in F12K medium (Procell), DU145 was cultivated in DMEM medium (Gibco), and 293T, LNCaP, and BPH-1 were maintained in RPMI-1640 medium (Gibco). All media listed above were supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco). All cell lines were maintained at 37°C with 5% CO₂. For the autophagy induction experiments, all stable or transient transfected cell lines were maintained in EBSS in the presence of 10 μM BAF and/or 50 nM CQ for 8 h. DMSO was used as NC.