C8661

For hypoxic experiments, OCCM-30 cells were cultivated supplemented with 100 μM or 400 μM cobalt (II) chloride hexahydrate (CoCl₂) for indicated time periods to mimick the different hypoxic culture conditions [28 (link)]. Briefly, CoCl₂ (C8661, Sigma-Aldrich, Taufirchen, Germany) was dissolved directly to the growth medium and sterilized through a sterile 0.2 μm spare membrane filter (Z333905-1EA, Merck, Darmstadt, Germany) to reach final concentrations of 100 μM or 400 μM. These concentrations are based on the hypoxic concentration established in previous studies [22 (link),28 (link)]. Cells cultured without CoCl₂ served as the normoxic control.

Cobalt (II) chloride hexahydrate (CoCl₂ 6H₂O, C8661, Sigma, United States) was dissolved in a 100 mM stock solution with sterilized water and stored at −80°C freezer. To find out the preferred concentration for hypoxic injury, different concentrations of CoCl₂ solution (0, 1, 10, 20, and 50 mM) were applied to zebrafish embryos every 12 h until 96 hpf. Survival rate was calculated under different concentration working solution every 12 h. WT and serpini1^−/− zebrafish were exposed to the preferred concentration for phenotype observation (survival rate≥50% at 96 hpf) according to the survival curve. Morphology phenotypes including hatch rate at 48hpf, survival rate at 96 hpf, teratogenic effects including pericardial edema, spine deformation, and abnormality of the head, eye (n = 200 per group) were analyzed by visual assessment under a dissection microscope (Olympus, DP73, Japan).

HUVECs were treated with 200 μg/mL cobalt(II) chloride hydrate (C8661, Sigma) for the indicated times. In control experiments, a comparable amount of vehicle (H₂O) was added.

To investigate the mechanism of action of downstream signalling pathways after si-FAM13A transfection, cobalt (II) chloride hexahydrate was used to chemically induce hypoxia and create a cellular hypoxic environment. Cobalt (II) chloride hexahydrate (C8661; Sigma-Aldrich, shanghai, China) treatment was performed after the Qinchuan bovine precursor adipocyte density reached 70–80%. The concentration and duration of action of the added cobalt (II) chloride hexahydrate were determined and total RNA and total protein were collected from treated cells.

Cell lines in this study include six human BCa lines (T24, UMUC3, J82, 5637, BIU87, and RT4), a human bladder epithelial cell line (SV-HUC-1), and a human embryonic kidney cell line (293 T). All cell lines were provided by the Stem Cell Bank, Chinese Academy of Sciences in Shanghai and have recently been authenticated. RPMI-1640 medium was used for SV-HUV-1, T24, 5637, BIU87 and RT4 cell culture, DMEM medium was applied for UMUC3 and 293 T cell culture, and MEM medium was utilized for J82 cell culture. All culture mediums were freshly prepared with 10% fetal bovine serum (FBS). To simulate the hypoxic conditions, cells were subjected to a 4-h pretreatment of CoCl2 (100 μM, Sigma c8661) prior to collection [36 (link), 37 (link)].

Cell lines from OCCC (ES2; CRL-1978) and HG-OSC (OVCAR-3; HTB-161) were obtained from American Type Culture Collection (ATCC). Cells were maintained at 37°C in a humidified 5% CO₂ atmosphere, and cultured in Dulbecco’s Modified Eagle medium (DMEM, 41965-039, Gibco, Life Technologies), containing 4.5 g/L of D-glucose and 0.58 g/L of L-glutamine, 1% FBS (S 0615, Merck), 1% antibiotic-antimycotic (P06-07300, PAN Biotech) and 0.1% gentamicin (15750-060, Gibco, Life Technologies). Cells were exposed either to 0.402 mM L-cysteine (102839, Merck) and/or to hypoxia induced with 0.1 mM cobalt chloride (CoCl₂) (C8661, Sigma-Aldrich) as previously (Nunes et al., 2018a (link),b (link)).
Prior to any experiment, cells were synchronized under starvation (culture medium without FBS) for 8 h at 37°C and 5% CO₂.

The glucose mineral medium for shake flasks was prepared in milliQ-H₂O (18.2 MΩ cm) by dissolving 11.2 g/l Na₂HPO₄–7H₂O (S9390, Sigma-Aldrich), 3 g/l KH₂PO₄ (P5655, Sigma-Aldrich), 0.5 g/l NaCl (27810.295, VWR), 1 g/l NH₄Cl (A9434, Sigma-Aldrich), 0.2465 g/l MgSO₄–7H₂O (M5921, Sigma-Aldrich), 4 g/l glucose (101176 K, VWR), 2 ml/l of a 50 mg/l CoCl₂–6H₂O solution (C8661, Sigma-Aldrich) and 2 ml/l medium of a trace element solution containing 10 g/l FeSO₄–7H₂O (F8633, Sigma-Aldrich), 2.25 g/l ZnSO₄–7H₂O (Z0251, Sigma-Aldrich), 2 g/l CaCl₂–2H₂O (223506, Sigma-Aldrich), 1 g/l CuSO₄–5H₂O (197722500, Fisher Scientific), 0.38 g/l MnCl₂–4H₂O (M5005, Sigma-Aldrich), 0.14 g/l H₂BO₃ (B6768, Sigma-Aldrich) and 0.1 g/l (NH₄)6Mo₇O₂₄–4H₂O (1011820250, Merck).